Agents for use in the treatment of alzheimer&#39;s disease

ABSTRACT

The invention relates to the identification of pharmacological agents to be used in the treatment of Alzheimer&#39;s disease and related pathological conditions and compositions for treatment of conditions caused by amyloidosis, Aβ-mediated formation of ROS, or both, such as Alzheimer&#39;s disease, are disclosed.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSOREDRESEARCH AND DEVELOPMENT

[0001] Part of the work performed during the development of thisinvention utilized U.S. Government Funds under Grant No. R29AG12686 fromthe National Institutes of Health. The government may have certainrights in this invention.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention is in the field of medicinal chemistry. Inparticular, the invention is related to compositions for treatment ofAlzheimer's disease.

[0004] 2. Related Art

[0005] Polymers of Abeta (Aβ), the 4.3 kD, 39-43 amino acid peptideproduct of the transmembrane protein, amyloid protein precursor (APP),are the main components extracted from the neuritic and vascular amyloidof Alzheimer's disease (AD) brains. Aβ deposits are usually mostconcentrated in regions of high neuronal cell death, and may be presentin various morphologies, including amorphous deposits, neurophil plaqueamyloid, and amyloid congophilic angiopathy (Masters, C. L., et al.,EMBO J. 4:2757 (1985); Masters, C. L. et al., Proc. Natl. Acad. Sci. USA82: 4245 (1985)). Growing evidence suggests that amyloid deposits areintimately associated with the neuronal demise that leads to dementia inthe disorder.

[0006] The presence of an enrichment of the 42 residue species of Aβ inthese deposits suggests that this species is more pathogenic. The 42residue form of Aβ (Aβ₁₋₄₂), while a minor component of biologicalfluids, is highly enriched in amyloid, and genetic studies stronglyimplicate this protein in the etiopathogenesis of AD. Amyloid depositsare decorated with inflammatory response proteins, but biochemicalmarkers of severe oxidative stress such as peroxidation adducts,advanced glycation end-products, and protein cross-linking are seen inproximity to the lesions. To date, the cause of Aβ deposits is unknown,although it is believed that preventing these deposits may be a means oftreating the disorder.

[0007] When polymers of Aβ are placed into culture with rat hippocampala neurons, they are neurotoxic (Kuo, Y -M., et al., J. Biol. Chem.271:4077-81 (1996); Roher, A. E., et al., Journal of BiologicalChemistry 271:20631-20635 (1996)). The mechanism underlying theformation of these neurotoxic polymeric Aβ species remains unresolved.The overexpression of Aβ alone cannot sufficiently explain amyloidformation, since the concentration of Aβ required for precipitation isnot physiologically plausible. That alterations in the neurochemicalenvironment are required for amyloid formation is indicated by itssolubility in neural phosphate buffer at concentrations of up to 16mg/ml (Tomski, S. & Murphy, R. M., Archives of Biochemistry andBiophysics 294:630 (1992)), biological fluids such as cerebrospinalfluid (CSF) (Shoji, M., et al., Science 258:126 (1992); Golde, T. E., etal. Science, 255(5045):728-730 (1992); Seubert, P., et al., Nature359:325 (1992); Haass, C., et al., Nature 359:322 (1992)) and in theplaque-free brains of Down's syndrome patients (Teller, J. K., et al.,Nature Medicine 2:93-95 (1996)).

[0008] Studies into the neurochemical vulnerability of Aβ to formamyloid have suggested altered zinc and [H⁺] homeostasis as the mostlikely explanations for amyloid deposition. Aβ is rapidly precipitatedunder mildly acidic conditions in vitro (pH 3.5-6.5) (Barrow, C. J. &Zagorski, M. G., Science 253:179-182 (1991); Fraser, P. E., et al.,Biophys. J. 60:1190-1201 (1991); Barrow, C. J., et al., J. Mol. Biol.225:1075-1093 (1992); Burdick, D., J. Biol. Chem. 267:546-554 (1992);Zagorski, M. G. & Barrow, C. J., Biochemistry 31:5621-5631 (1992);Kirshenbaum, K. & Daggett, V., Biochemistry 34:7629-7639 (1995); Wood,S. J., et al., J. Mol. Biol. 256:870-877 (1996)). Recently, it has beenshown that the presence of certain biometals, in particular redoxinactive Zn²⁺ and, to a lesser extent, redox active Cu²⁺ and Fe³⁺,markedly increases the precipitation of soluble Aβ (Bush, A. I., et al,J. Biol. Chem. 268:16109 (1993); Bush, A. I., et al., J. Biol. Chem.269:12152(1994); Bush, A. I., et al., Science 265:1464(1994); Bush, A.I., et al., Science 268:1921 (1995)). At physiological pH, Aβ₁₋₄₀specifically and saturably binds Zn²⁺, manifesting high affinity binding(KD=107 nM) with a 1:1 (Zn²⁺:Aβ) stoichiometry, and low affinity binding(KD=5.2 μM) with a 2:1 stoichiometry.

[0009] The reduction by APP of copper (II) to copper (I) may lead toirreversible Aβ aggregation and SDS-resistant polymerization. Thisreaction may promote an environment that would enhance the production ofhydroxyl radicals, which may contribute to oxidative stress in AD(Multhaup, G., et al., Science 271: 1406-1409 (1996)). A precedence forabnormal Cu metabolism already exists in the neurodegenerative disordersof Wilson's disease, Menkes' syndrome and possibly familial amyotrophiclateral sclerosis (Tanzi, R. E. et al., Nature Genetics 5:344 (1993);Bull, P. C., et al., Nature Genetics 5:327 (1993); Vulpe, C., et al.,Nature Genetics 3:7 (1993); Yamaguchi, Y., et al., Biochem. Biophys.Res. Commun. 197:271 (1993); Chelly, J., et al., Nature Genetics 3:14(1993); Wang, D. & Munoz, D. G., J. Neuropathol. Exp. Neurol. 54:548(1995); Beckman, J. S., et al., Nature 364:584 (1993); Hartmann, H. A. &Evenson, M. A., Med. Hypotheses 38:75 (1992)).

[0010] Although much fundamental pathology, genetic susceptibility andbiology associated with AD is becoming clearer, a rational chemical andstructural basis for developing effective drugs to prevent or cure thedisease remains elusive. While the genetics of the disorder indicatesthat the metabolism of Aβ is intimately associated with theetiopatholgenesis of the disease, drugs for the treatment of AD have sofar focused on “cognition enhancers” which do not address the underlyingdisease processes.

SUMMARY OF THE INVENTION

[0011] An aspect of the present invention contemplates a method fortreating Alzheimer's disease (AD) in a subject, said method comprisingadministering to said subject an effective amount of an agent which iscapable of inhibiting or otherwise reducing metal-mediated production offree radicals.

[0012] The present invention provides a method for treating AD in asubject, said method comprising administering to said subject aneffective amount of an agent comprising a metal chelator and/or a metalcomplexing compound for a time and under conditions sufficient toinhibit or otherwise reduce metal-mediated production of free radicalsby Aβ.

[0013] In one aspect, the free radicals are reactive oxygen species suchas O₂ or OH. In another aspect, the free radicals include forms of Aβ.

[0014] The agent of this aspect of the present invention may contain oneor more than one compound such as a metal chelator or metal complexingcompound such as but not limited to DTPA, bathocuproine,bathophenanthroline, clioquinol, penicillamine, or derivatives,homologues or analogues thereof. Alternatively, or in addition, theagent may comprise an antioxidant or other molecule capable ofinterfering with Aβ peptide-mediated radical formation.

[0015] One aspect of the present invention comprises an agent for use intreating AD in a subject comprising a metal chelator, metal complexingcompound and/or a compound capable of inhibiting free radical formationby interaction of Aβ peptides and biometals, said agent optionallyfurther comprising one or more pharmaceutically acceptable carriersand/or diluents.

[0016] In one aspect, the invention relates to a method of treatingamyloidosis in a subject, said method comprising administering to saidsubject a combination of (a) a metal chelator selected from the groupconsisting of: bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA,penicillamine, TETA, and TPEN, or hydrophobic derivatives thereof; and(b) clioquinol, for a time and under conditions to bring about saidtreatment; wherein said combination reduces, inhibits or otherwiseinterferes with Aβ-mediated production of radical oxygen species.

[0017] In a preferred embodiment, the metal chelator is bathocuproine.

[0018] In another aspect, said method further comprises administering asupplement selected from the group consisting of: ammonium salt, calciumsalt, magnesium salt, and sodium salt.

[0019] In a preferred embodiment, the supplement is magnesium salt.

[0020] In another aspect, said method further comprises administering tothe subject an effective amount of a compound selected from the groupconsisting of: rifampicin, disulfiram, and indomethacin, or apharmaceutically acceptable salt thereof.

[0021] In yet another aspect, the invention relates to a method oftreating amyloidosis in a subject, said method comprising administeringto said subject an effective amount of a combination of (a) a salt of ametal chelator, wherein said chelator is selected from the groupconsisting of: bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA,penicillamine, TETA, and TPEN, or hydrophobic derivatives thereof, and(b) clioquinol; wherein said salt of the metal chelator is selected fromthe group consisting of: ammonium, calcium, magnesium, and sodium; andwherein said combination reduces, inhibits or otherwise interferes withAβ-mediated production of radical oxygen species.

[0022] In a preferred embodiment, the metal chelator is bathocuproine.

[0023] In another preferred embodiment, the salt of the metal chelatoris a magnesium salt.

[0024] In another aspect, said method further comprises administering tothe subject an effective amount of a compound selected from the groupconsisting of: rifampicin, disulfiram, and indomethacin, or apharmaceutically acceptable salt thereof.

[0025] In yet another aspect, the invention relates to a method oftreating amyloidosis in a subject, said method comprising administeringto said subject an effective amount of a combination of (a) a chelatorspecific for copper, and (b) clioquinol; wherein said combinationreduces, inhibits or otherwise interferes with Aβ-mediated production ofradical oxygen species.

[0026] In a preferred embodiment, the chelator specific for copper isspecific for the reduced form of copper. Most preferrably, the chelatoris bathocuproine or a hydrophobic derivative thereof.

[0027] In yet another aspect, the invention relates to a method oftreating amyloidosis in a subject, said method comprising administeringto said subject an effective amount of a combination of (a) analkalinizing agent and (b) clioquinol; wherein said combination reduces,inhibits or otherwise interferes with Aβ-mediated production of radicaloxygen species.

[0028] In a preferred embodiment, the alkalinizing agent is magnesiumcitrate. In another preferred embodiment, the alkalinizing agent iscalcium citrate.

[0029] Still another aspect of the present invention contemplates amethod of treating AD in a subject comprising administering to saidsubject an agent capable of promoting, inducing or otherwisefacilitating resolubilization of Aβ deposits in the brain for a time andunder conditions to effect said treatment.

[0030] In yet another aspect, the invention relates to a method oftreating amyloidosis in a subject, said method comprising administeringto said subject a combination of (a) a metal chelator selected from thegroup consisting of: bathocuproine, bathophenanthroline, DTPA, EDTA,EGTA, penicillamine, TETA, and TPEN, or hydrophobic derivatives thereof;and (b) clioquinol, for a time and under conditions to bring about saidtreatment; wherein said combination prevents formation of Aβ amyloid,promotes, induces or otherwise facilitates resolubilization of Aβdeposits, or both.

[0031] In a preferred embodiment, the metal chelator is bathocuproine.

[0032] In another aspect, said method further comprises administering asupplement selected from the group consisting of: ammonium salt, calciumsalt, magnesium salt, and sodium salt.

[0033] In a preferred embodiment, the supplement is magnesium salt.

[0034] In another aspect, said method further comprises administering tothe subject an effective amount of a compound selected from the groupconsisting of: rifampicin, disulfiram, and indomethacin, or apharmaceutically acceptable salt thereof.

[0035] In yet another aspect, the invention relates to a method oftreating amyloidosis in a subject, said method comprising administeringto said subject an effective amount of a combination of (a) a salt of ametal chelator, wherein said chelator is selected from the groupconsisting of: bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA,penicillamine, TETA, and TPEN, or hydrophobic derivatives thereof, and(b) clioquinol; wherein said salt of the metal chelator is selected fromthe group consisting of: ammonium, calcium, magnesium, and sodium; andwherein said combination prevents formation of Aβ amyloid, promotes,induces or otherwise facilitates resolubilization of Aβ deposits, orboth.

[0036] In a preferred embodiment, the metal chelator is bathocuproine.In another preferred embodiment, the salt of the metal chelator is amagnesium salt.

[0037] In another aspect, said method further comprises administering tothe subject an effective amount of a compound selected from the groupconsisting of: rifampicin, disulfiram, and indomethacin, or apharmaceutically acceptable salt thereof.

[0038] In yet another aspect, the invention relates to a method oftreating amyloidosis in a subject, said method comprising administeringto said subject an effective amount of a combination of (a) a chelatorspecific for copper, and (b) clioquinol; wherein said combinationprevents formation of Aβ amyloid, promotes, induces or otherwisefacilitates resolubilization of Aβ deposits, or both.

[0039] In a preferred embodiment, the chelator specific for copper isspecific for the reduced form of copper. Most preferrably, the chelatoris bathocuproine or a hydrophobic derivative thereof.

[0040] In yet another aspect, the invention relates to a method oftreating amyloidosis in a subject, said method comprising administeringto said subject an effective amount of a combination of (a) analkalinizing agent and (b) clioquinol; wherein said combination preventsformation of Aβ amyloid, promotes, induces or otherwise facilitatesresolubilization of Aβ deposits, or both.

[0041] In a preferred embodiment, the alkalinizing agent is magnesiumcitrate. In another preferred embodiment, the alkalinizing agent iscalcium citrate.

[0042] Still another aspect of the invention relates to a pharmaceuticalcomposition for treatment of conditions caused by amyloidosis,Aβ-mediated ROS formation, or both, comprising: (a) a metal chelatorselected from the group consisting of: bathocuproine,bathophenanthroline, DTPA, EDTA, EGTA, penicillamine, TETA, and TPEN, orhydrophobic derivatives thereof; and (b) clioquinol, together with oneor more pharmaceutically acceptable carriers or diluents.

[0043] In a preferred embodiment, the metal chelator is bathocuproine.

[0044] In another aspect, said method further comprises administering asupplement selected from the group consisting of: ammonium salt, calciumsalt, magnesium salt, and sodium salt.

[0045] In a preferred embodiment, the supplement is magnesium salt.

[0046] In another aspect, said composition further comprises a compoundselected from the group consisting of: rifampicin, disulfiram, andindomethacin, or a pharmaceutically acceptable salt thereof.

[0047] In yet another aspect, the invention relates to a pharmaceuticalcomposition for treatment of conditions caused by amyloidosis,Aβ-mediated ROS formation, or both, comprising a combination of (a) asalt of a metal chelator selected from the group consisting of:bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, penicillamine,TETA, and TPEN, or hydrophobic derivatives thereof; and (b) clioquinol;wherein said salt of the metal chelator is selected from the groupconsisting of: ammonium, calcium, magnesium, and sodium, together withone or more pharmaceutically acceptable carriers or diluents.

[0048] In a preferred embodiment, the metal chelator is bathocuproine.In another preferred embodiment, the salt of the metal chelator is amagnesium salt.

[0049] In another aspect, said composition further comprises a compoundselected from the group consisting of: rifampicin, disulfiram, andindomethacin, or a pharmaceutically acceptable salt thereof.

[0050] In yet another aspect, the invention relates to a pharmaceuticalcomposition for treatment of conditions caused by amyloidosis,Aβ-mediated ROS formation, or both, comprising a chelator specific forcopper, with one or more pharmaceutically acceptable carriers ordiluents.

[0051] In a preferred embodiment, the chelator specific for copper isspecific for the reduced form of copper. Most preferrably, the chelatoris bathocuproine or a hydrophobic derivative thereof.

[0052] In yet another aspect, the invention relates to a pharmaceuticalcomposition for treatment of conditions caused by amyloidosis,Aβ-mediated ROS formation, or both, comprising a combination of (a) analkalinizing agent and (b) clioquinol; together with one or morepharmaceutically acceptable carriers or diluents.

[0053] In a preferred embodiment, the alkalinizing agent is magnesiumcitrate. In another preferred embodiment, the alkalinizing agent iscalcium citrate.

[0054] Still another aspect of the invention relates to a composition ofmatter comprising: (a) a metal chelator selected from the groupconsisting of: bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA,penicillamine, TETA, and TPEN, or hydrophobic derivatives thereof; and(b) clioquinol.

[0055] In a preferred embodiment, the metal chelator is bathocuproine.

[0056] In another aspect, said method further comprises administering asupplement selected from the group consisting of: ammonium salt, calciumsalt, magnesium salt, and sodium salt.

[0057] In a preferred embodiment, the supplement is magnesium salt.

[0058] In another aspect, said composition further comprises aneffective amount of a compound selected from the group consisting of:rifampicin, disulfiram, and indomethacin, or a pharmaceuticallyacceptable salt thereof.

[0059] In yet another aspect, the invention relates to a composition ofmatter comprising a combination of (a) a salt of a metal chelatorselected from the group consisting of: bathocuproine,bathophenanthroline, DTPA, EDTA, EGTA, penicillamine, TETA, and TPEN, orhydrophobic derivatives thereof; and (b) clioquinol; wherein said saltof the metal chelator is selected from the group consisting of:ammonium, calcium, magnesium, and sodium.

[0060] In a preferred embodiment, the metal chelator is bathocuproine.In another preferred embodiment, the salt of said metal chelator is amagnesium salt.

[0061] In another aspect, said composition further comprises aneffective amount of a compound selected from the group consisting of:rifampicin, disulfiram, and indomethacin, or a pharmaceuticallyacceptable salt thereof.

[0062] In yet another aspect, the invention relates to a composition ofmatter comprising a combination of (a) an alkalinizing agent and (b)clioquinol.

BRIEF DESCRIPTION OF THE FIGURES

[0063]FIG. 1 is a graph showing the proportion of soluble Aβ₁₋₄₀remaining following centrifugation of reaction mixtures.

[0064] FIGS. 2A-2C:

[0065]FIG. 2A is a graph showing the proportion of soluble Aβ₁₋₄₀remaining in the supernatant after incubation with various metal ions.

[0066]FIG. 2B is a graph showing a turbidometric analysis of pH effecton metal ion-induced Aβ₁₋₄₀ aggregation.

[0067]FIG. 2C is a graph showing the proportion of soluble Aβ₁₋₄₀remaining in the supernatant after incubation with various metal ions,where high metal ion concentrations were used.

[0068]FIG. 3 is a graph showing a competition analysis of Aβ₁₋₄₀ bindingto Cu²⁺.

[0069] FIGS. 4A-4C:

[0070]FIG. 4A is a graph showing the proportion of soluble Aβ₁₋₄₀remaining in the supernatant following incubation at various pHs inPBS±Zn²⁺ or Cu²⁺.

[0071]FIG. 4B is a graph showing the proportion of soluble Aβ₁₋₄₀remaining in the supernatant following incubation at various pHs withdifferent Cu²⁺ concentrations.

[0072]FIG. 4C is a graph showing the relative aggregation of nMconcentrations of Aβ₁₋₄₀ at pH 7.4 and 6.6 with different Cu²⁺concentrations.

[0073] FIGS. 5A and 5B:

[0074]FIG. 5A is a graph showing a turbidometric analysis ofCu²⁺-induced Aβ₁₋₄₀ aggregation at pH 7.4 reversed by successive cyclesof chelator.

[0075]FIG. 5B is a graph showing a turbidometric analysis of thereversibility of Cu²⁺-induced Aβ₁₋₄₀ aggregation as the pH cyclesbetween 7.4 and 6.6.

[0076]FIG. 6 shows the amino acid sequence of APP₆₆₉₋₇₁₆ near Aβ₁₋₄₂.Rat Aβ is mutated (R5G, Y10F, H13R; bold). Possible metal-bindingresidues are underlined.

[0077]FIG. 7 is a graph showing the effects of pH, Zn²⁺ or Cu²⁺ upon Aβdeposit formation.

[0078]FIG. 8 is a western blot showing the extraction of Aβ frompost-mortem brain tissue.

[0079]FIG. 9 is a western blot showing Aβ SDS-resistant polymerizationby copper.

[0080]FIG. 10 is a graph showing Cu⁺ generation by Aβ.

[0081]FIG. 11 is a graph showing H₂O₂ production by Aβ.

[0082]FIG. 12 is a graphical representation showing a model for thegeneration of reduced metal ions, O₂ ⁻, H₂O₂, and OH. by Aβ peptides.Note that Aβ facilitates two consecutive steps in the pathway: thereduction of metal ions, and the reaction of O₂ with reduced metal ions.The peptide does not appear to be consumed or modified in a one hourtime frame by participation in these reactions.

[0083]FIGS. 13A and 13B are graphical representations showing Fe³⁺ orCu²⁺ reduction by Aβ peptides.

[0084]FIG. 13A illustrates the reducing capacity of Aβ species (10 μM),compared to Vitamin C and insulin (Sigma) (all 10 μM) towards Fe³⁺ orCu²⁺ (10 μM) in PBS, pH 7.4, after 1 hour co-incubation, 37° C. Dataindicate concentration of reduced metal ions generated.

[0085]FIG. 13B shows the effect of oxygen tension and chelation uponAβ₁₋₄₂ metal reduction. Aβ₁₋₄₂ was incubated as in FIG. 13A undervarious buffer gas conditions. “Ambient”=no efforts were made to adjustthe gas tension in the bench preparations of the buffer vehicle,“O₂”=100% O₂ was continuously bubbled through the PBS vehicle for 2hours (at 20° C.), before the remainder of the incubation componentswere added, “Ar”=100% Ar was continuously bubbled through the PBSvehicle for 2 hours (at 20° C.), before the remainder of the incubationcomponents were added. “+DFO or TETA”=Desferrioxamine (DFO, Sigma, 200μM) was added to the Aβ₁₋₄₂ incubation in the presence of Fe³⁺ 10 μM, ortriethylenetetramine dihydrochloride (TETA, Sigma, 200 μM) was added tothe Aβ₁₋₄₂ incubation in the presence of Cu²⁺ 10 μM, under ambientoxygen conditions. All data points are means ±SD, n=3.

[0086] FIGS. 14A-14E are graphical representations showing production ofH₂O₂ from the incubation of Aβ in the presence of substoichiometricamounts of Fe³⁺ or Cu²⁺.

[0087]FIG. 14A shows H₂O₂ produced by Aβ₁₋₄₂ (in PBS, pH 7.4, underambient gas conditions, 1 hour, 37° C.) following co-incubation withvarious concentrations of catalase in the presence of 1 μM Fe³⁺.

[0088]FIG. 14B shows a comparison of H₂O₂ generation by variant Aβspecies: Aβ₁₋₄₂, Aβ₁₋₄₀, rat Aβ₁₋₄₀, Aβ₄₀₋₁, and Aβ₁₋₂₈, (vehicleconditions as in FIG. 14A).

[0089]FIG. 14C shows the effect of metal chelators (200 μM) on H₂O₂production from Aβ₁₋₄₂ when incubated in the presence of Fe³⁺ or Cu²⁺ (1μM) (vehicle conditions as in FIG. 14A). BC=Bathocuproinedisulfonate,BP=Bathophenanthrolinedisulfonate. The effects of DFO were assessed inthe presence of Fe³⁺, and TETA was assessed in the presence of Cu²⁺, asindicated.

[0090]FIG. 14D shows H₂O₂ produced by Aβ₁₋₄₂, Aβ₁₋₄₀, and Vitamin C inthe presence of Fe³⁺ (1 μM) (in PBS, pH 7.4 buffer, 1 hr, 37° C.) undervarious dissolved gas conditions (described in FIG. 13B): ambient air,O₂ enrichment, and anaerobic (Ar) conditions, as indicated.

[0091]FIG. 14E shows H₂O₂ produced by Aβ₁₋₂, Aβ₁₋₄₀, and Vitamin C inthe presence of Cu²⁺ (1 μM) (in PBS, pH 7.4 buffer, 1 hr, 37° C.) undervarious dissolved gas conditions (as in FIG. 14D). All data points aremeans ±SD, n=3.

[0092]FIG. 15A and 15B are graphical representations showing superoxideanion detection.

[0093]FIG. 15A shows the spectrophotometric absorbance at 250 nm (aftersubtracting buffer blanks) for Aβ₁₋₄₂ (10 μM, in PBS, pH 7.4, with 1 μMFe³⁺, incubated 1 hr, 37° C.) underambientair (+100 U/mL superoxidedismutase, SOD), O₂ enrichment, and anaerobic (Ar). buffer gasconditions (described in FIG. 13B).

[0094]FIG. 15B shows the spectrophotometric absorbance at 250 nm (aftersubtracting buffer blanks) for variant Aβ peptides: Aβ₁₋₄₂, Aβ₁₋₄₀, ratAβ₁₋₄₀, Aβ₄₀₋₁, and Aβ₁₋₂₈ (10 μM in PBS, pH 7.4, with 1 μM Fe³⁺,incubated 1 hr, 37° C., under ambient buffer gas conditions). All datapoints are means ±SD, n=3.

[0095]FIG. 16A and 16B are graphical representations showing productionof the hydroxyl radical (OH.) from the incubation of Aβ in the presenceof substoichiometric amounts of Fe³⁺ or Cu²⁺.

[0096]FIG. 16A shows the signal from the TBARS assay of OHS producedfrom Vitamin C (100 μM) and variant Aβ species (10 μM): Aβ₁₋₄₂, Aβ₁₋₄₀,rat Aβ₁₋₄₀, Aβ₄₀₋₁, and Aβ₁₋₂₈ (in PBS, pH 7.4, with 1 μM Fe³⁺ or Cu²⁺as indicated, incubated 1 hr, 37° C., under ambient buffer gasconditions).

[0097]FIG. 16B illustrates the effect of OH.-specific scavengers uponOH. generation by Vitamin C and Aβ₁₋₄₂. Mannitol (5 mM, Sigma) ordimethyl sulfoxide (DMSO, 5 mM, Sigma), was co-incubated with Vitamin C(10 μM+500 μM H₂O₂) or Aβ₁₋₄₂ (10 μM) (conditions as for FIG. 16A). Alldata points are means ±SD, n=3.

[0098]FIG. 17 shows the reversibility of zinc-induced Aβ₁₋₄₀ aggregationwith EDTA. Aggregation induced by pH 5.5 was not reversable in the samemanner.

[0099]FIG. 18 shows the reversibility of zinc-induced aggregation ofAβ₁₋₄₀ mixed with 5% Aβ₁₋₄₂.

[0100] FIGS. 19A-19C show dilution curves for TPEN, EGTA, andbathocuproine, respectively, used in extracting a representative ADbrain sample. FIGS. 19A-19C show that metal chelators promote thesolubilization of Aβ from human brain sample homogenates.

[0101]FIGS. 20A and 20B

[0102]FIG. 20A shows a western blot of chelation response in a typicalAD brain.

[0103]FIG. 20B shows a western blot comparing extracted Aβ from an ADbrain (AD) to that of sedimentable deposits from healthy brain tissue(young control—C). In the experiments of

[0104]FIG. 20B, TBS buffer was used rather than PBS.

[0105]FIG. 21 shows an indicative blot from AD brain extract. The blotshows that chelation treatment results in disproportionatesolubilization of Aβ dimers, while PBS alone does not.

[0106]FIG. 22 shows that recovery of total soluble protein is notaffected by the presence of chelators in the homogenization step.

[0107]FIG. 23 is a graphical representation of resolubilization of Zn,Cu, or pH induced aggregates in vitro. Values are expressed as apercentage of Aβ signal after washing with TBS alone.

[0108]FIG. 24 shows extraction of Aβ from brain tissue with clioquinol.Undiluted (100%) clioquinol is 1.6 μM. S1 and S2 represent twosequential extractions from AD-affected tissue.

[0109] FIGS. 25A and 25B:

[0110]FIG. 24A shows a western blot of Aβ extracted from brain tissue byvarious concentrations of clioquinol.

[0111]FIG. 24B is a graphic representation of solubilization of Aβ byclioquinol.

[0112]FIG. 26 is a graph showing the proportion of total Aβ extractedutilizing PBS buffer alone, clioquinol (CQ), bathocuproine (BC), orclioquinol together with bathocuproine (CQ+BC).

[0113]FIG. 27 shows that extraction volume affects Aβ solubilisation.

[0114]FIGS. 28A and 28B

[0115]FIG. 28A shows the effect of metals upon the solubility ofbrain-derived Aβ: copper and zinc can inhibit the solubilization of Aβ.

[0116]FIG. 28B shows that Aβ solubility in metal-depleted tissue isrestored by supplementing with magnesium.

[0117]FIGS. 29A and 29B

[0118]FIG. 29A shows that patterns of chelator-promoted solubilisationof Aβ differ in AD and aged-matched, non-AD tissue.

[0119] Upper panel: representative blot from AD specimen.

[0120] Lower panel: representative blot from aged non-AD tissue bearinga similar total Aβ load.

[0121]FIG. 29B shows soluble Aβ resulting from chelation treatment forAD and aged-matched, non-AD tissue, expressed as a percentage of thePBS-only treatment group.

[0122]FIG. 30 shows that chelation promotes the solubilization of Aβ₁₋₄₀and Aβ₁₋₄₂ from AD and non-AD tissue. Representative AD (left panels)and aged-matched control specimens (right panels) were prepared asdescribed in PBS or 5 mM BC. Identical gels were run and Western blotswere probed with mAbs WO2 (raised against residues 5-16, recognizesAβ₁₋₄₀ and Aβ₁₋₄₂) G210 (raised against residues 35-40, recognizesAβ₁₋₄₀) or G211 (raised against residues 35-42, recognizes Aβ₁₋₄₂) (SeeIda, N., et al., J. Biol. Chem., 271:22908 (1996)).

[0123]FIG. 31A and 31B

[0124]FIG. 31A shows SDS-resistant polymerization of human Aβ₁₋₄₀ versushuman Aβ₁₋₄₂ with Zn²⁺ or Cu²⁺.

[0125]FIG. 31B shows SDS-resistant polymerization of rat Aβ₁₋₄₀ withCu²⁺ or Fe³⁺.

[0126] FIGS. 32A-32C

[0127]FIG. 32A shows H₂O₂/Cu induced SDS-resistant polymerization ofAβ₁₋₄₂ (2.5 μM).

[0128]FIG. 32B shows H₂O₂/Fe induced SDS-resistant polymerization ofAβ₁₋₄₂ (2.5 μM).

[0129]FIG. 32C shows that BC attenuates SDS-resistant polymerization ofAβ₁₋₄₂ (2.5 μM).

[0130]FIGS. 33A and 33B show that H₂O₂ generation is required forSDS-resistant polymerization of human Aβ₁₋₄₂. Solution concentrations ofmetal ion and H₂O₂ were 30 μM and 100 μM, respectively.

[0131]FIG. 33A shows that TCEP (Tris(2-Carboxyethyl)-PhosphineHydrochloride) attenuates SDS-resistant Aβ₁₋₄₂ polymerization. Aβ₁₋₄₂(2.5 μM), H₂O₂ (100 μM), ascorbic acid (100 μM), TCEP (100 μM).

[0132]FIG. 33B shows that anoxic conditions prevent SDS-resistant Aβpolymerization. Aβ₁₋₄₂ (2.5 μM) was incubated with no metal or Cu²⁺ ateither pH 7.4 or 6.6 and incubated for 60 min. at 25° C. under normal orargon purged conditions. Argon was continuously bubbled through thebuffer for 2 h (at 20° C.) before the remainder of the incubationcomponents were added.

[0133] FIGS. 34A-34E show dissolution of SDS-resistant Aβ polymers.

[0134]FIG. 34A shows that chaotrophic agents are unable to disruptpolymerization.

[0135]FIG. 34B shows that metal ion chelators disrupt SDS-resistantAβ₁₋₄₀ polymers.

[0136]FIG. 34C shows that metal ion chelators disrupt SDS-resistantAβ₁₋₄₂ polymers. The chelators, their log stability constant, and theirmolecular weight, respectively, are as follows: TETA(tetraethylenediamine), 20.4, 146; EDTA (ethylenediaminetetra aceticacid), 18.1, 292; DTPA (diethylenetriaminopenta acetic acid), 21.1, 393;CDTA (trans-1,2-diaminocyclohexanetetra acetic acid), 22.0, 346; and NTA(nitrilotriacetic acid), 13.1, 191.

[0137]FIG. 34D shows that α-helical promoting solvents and low pHdisrupt polymers. Aliquots of Aβ₁₋₄₂ were incubated at pH 1 or withDMSO/HFIP (75%:25%) for 2 h (30 min., 37° C.).

[0138]FIG. 34E shows that metal ion chelators disrupt SDS-resistant Aβpolymers extracted from AD brains. Aliquots of SDS-resistant Aβ polymersextracted from AD brains were incubated with no chelator, TETA (1 mM or5 mM) or BC (1 mM or 5 mM) for 2 h (30 min., 37° C.) and aliquotscollected for analysis. Monomer Aβ₄₀ is indicated.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0139] Definitions

[0140] In the description that follows, a number of terms are utilizedextensively. In order to provide a clear and consistent understanding ofthe specification and claims, including the scope to be given suchterms, the following definitions are provided.

[0141] Aβ peptide is also known in the art as Aβ, P protein, β-A4 andA4. In the present invention, the Aβ peptide may be comprised ofpeptides Aβ₁₋₃₉ Aβ₁₋₄₀ Aβ₁₋₄₁, Aβ₁₋₄₂, and Aβ₁₋₄₃. The most preferredembodiment of the invention makes use of Aβ₁₋₄₀. However, any of the Aβpeptides may be employed according to the present invention. Aβ peptidesof the invention include Aβ_(X-39), Aβ_(X-40), Aβ_(X-42), and Aβ_(X-43),where X is less than or equal to 17; and Aβ_(Y-17), where Y is less thanor equal to 5. The sequence of Aβ peptide is found in Hilbich, C., etal., J. Mol. Biol. 228:460-473 (1992).

[0142] Amyloid as is commonly known in the art, and as is intended inthe present specification, is a form of aggregated protein.

[0143] Amyloidosis is any disease characterized by the extracellularaccumulation of amyloid in various organs and tissues of the body.

[0144] Aβ Amyloid is an aggregated Aβ peptide. It is found in the brainsof patients afflicted with AD and DS and may accumulate following headinjuries.

[0145] Biological fluid means fluid obtained from a person or animalwhich is produced by said person or animal. Examples of biologicalfluids include but are not limited to cerebrospinal fluid (CSF), blood,serum, and plasma. In the present invention, biological fluid includeswhole or any fraction of such fluids derived by purification by anymeans, e.g., by ultrafiltration or chromatography.

[0146] Copper(II), unless otherwise indicated, means salts of Cu²⁺,i.e., Cu²⁺ in any form, soluble or insoluble.

[0147] Copper(I), unless otherwise indicated, means salts of Cu⁺, i.e.,Cu⁺ in any form, soluble or insoluble.

[0148] Metal chelators include metal-binding molecules characterized bytwo or more polar groups which participate in forming a complex with ametal ion, and are generally well-known in the art for their ability tobind metals competitively.

[0149] Physiological solution as used in the present specification meansa solution which comprises compounds at physiological pH, about 7.4,which closely represents a bodily or biological fluid, such as CSF,blood, plasma, et cetera.

[0150] Treatment: delay or prevention of onset, slowing down or stoppingthe progression, aggravation, or deterioration of the symptoms and signsof Alzheimer's disease, as well as amelioration of the symptoms andsigns, or curing the disease by reversing the physiological andanatomical damage.

[0151] Zinc, unless otherwise indicated, means salts of zinc, i.e., Zn²⁺in any form, soluble or insoluble.

[0152] Methods for Identifying Agents Useful in the Treatment of AD

[0153] The aim of the present invention is to clarify both the factorswhich contribute to the neurotoxicity of Aβ polymers and the mechanismwhich underlies their formation. These findings can then be used to (i)identify agents that can be used to decrease the neurotoxicity of Aβ, aswell as the formation of Aβ polymers, and (ii) utilize such agents todevelop methods of preventing, treating or alleviating the symptoms ofAD and related disorders.

[0154] The present invention relates to the unexpected discovery that Aβpeptides directly produce oxidative stress through the generation ofabundant reactive oxygen species (ROS), which include hydroxyl radical(OH.) and hydrogen peroxide (H₂O₂). The production of ROS occurs by ametal (Cu, Fe) dependant, pH mediated mechanism, wherein the reductionof Cu²⁺ to Cu⁺, or Fe³⁺ to Fe²⁺, is catalyzed by Aβ. Aβ is highlyefficient at reducing Cu²⁺ and Fe³⁺.

[0155] All the redox properties of Aβ₁₋₄₀ (the most abundant form ofsoluble Aβ) are exaggerated in Aβ₁₋₄₂. Additionally, Aβ₁₋₄₂, but notAβ₁₋₄₀, recruits O₂ into spontaneous generation of another ROS, O₂ ⁻,which also occurs in a metal-dependent manner. The exaggerated redoxactivity of Aβ₁₋₄₂ and its enhanced ability to generate ROS are likelyto be the explanation for its neurotoxic properties. Interestingly, therat homologue of Aβ, which has 3 substitutions that have been shown toattenuate zinc binding and zinc-mediated precipitation, also exhibitsless redox activity than its human counterpart. This may explain why therat is exceptional in that it is the only mammal that does not exhibitamyloid pathology with age. All other mammals analyzed to date possessthe human Aβ sequence.

[0156] The sequence of ROS generation by Aβ follows the pathway ofsuperoxide-dismutation, which leads to hydrogen peroxide production in aCu/Fe-dependent manner. After forming H₂O₂, the hydroxyl radical (OH.)is rapidly formed by a Fenton reaction with the Fe or Cu that ispresent, even when these metals are only at trace concentrations. TheOH. radical is very reactive and rapidly attacks the Aβ peptide, causingit to polymerize. This is very likely to be the chemical mechanism thatcauses the SDS-resistant polymerization that is seen in mature plaqueamyloid. Importantly, the redox activity of Aβ is not attenuated byprecipitation of the peptide, suggesting that, in vivo, amyloid depositscould be capable of generating ROS in situ on an enduring basis. Thissuggests that the major source of the oxidative stress in an AD-affectedbrain are amyloid deposits.

[0157] A model for free radical and amyloid formation in AD is shown inFIG. 12. The proposed mechanism is explained as follows.

[0158] (1) Soluble and precipitated Aβ species possess superoxidedismutase (SOD)-like activity. Superoxide (O₂), the substrate for thedismutation, is generated both by spillover from mitochondrialrespiratory metabolism, and by Aβ₁₋₄₂ itself. Aβ-mediated dismutationproduces hydrogen peroxide (H₂O₂)(see FIG. 11), requiring Cu²⁺ or Fe³⁺,which are reduced during the reaction. Since H⁺ is required for H₂O₂production, an acidotic environment will increase the reaction.

[0159] (2) H₂O₂ is relatively stable, and freely permeable across cellmembranes. Normally, it will be broken down by intercellular catalase orglutathione peroxidase.

[0160] (3) In aging and AD, levels of H₂O₂ are high, and catalase andperoxidase activities are low. If H₂O₂ is not completely catalyzed, itwill react with reduced Cu⁺ and Fe²⁺ in the vicinity of Aβ to generatethe highly reactive hydroxyl radical (OH.) by Fenton chemistry.

[0161] (4) OH. engenders a non-specific stress and inflammatory responsein local tissue. Among the neurochemicals that are released frommicroglia and possibly neurons in the response are Zn²⁺, Cu²⁺ andsoluble Aβ. Familial AD increases the likelihood that Aβ₁₋₄₂ will bereleased at this point. Local acidosis is also part of thestress/inflammatory response. These factors combine to make Aβprecipitate and accumulate, presumably so that it may function in situas an SOD, since these factors induce reversible aggregation. Hence,more soluble Aβ species decorate the perimeter of the accumulatingplaque deposits.

[0162] (5) If Aβ encounters OH., it will cause covalently cross-linkingduring the oligomerization process, making it a more difficultaccumulation to resolubilize, and leading to the formation ofSDS-resistant oligomers characteristic of plaque amyloid.

[0163] (6) If Aβ₁₋₄₂ accumulates, it has the property of recruiting O₂as a substrate for the abundant production of O₂ ⁻ by a process that isstill not understood. Since O₂ is abundant in the brain, Aβ₁₋₄₂ isresponsible for setting off a vicious cycle in which the accumulation ofcovalently linked Aβ is a product of the unusual ability of Aβ to reduceO₂, and feed an abundant substrate (O₂ ⁻) to itself for dismutation,leading to OH. formation. The production of abundant free radicals bythe accumulating amyloid may further damage many systems including metalregulatory proteins, thus compounding the problem. This suggests thatthe major source of the oxidative stress in an AD-affected brain areamyloid deposits.

[0164] The metal-dependent chemistry of Aβ-mediated superoxidedismutation is reminiscent of the activity of superoxide dismutase(SOD). Interestingly, mutations of SOD cause amyotrophic lateralsclerosis, another neurodegenerative disorder. SOD is predominantlyintracellular, whereas Aβ is constitutively found in the extracellularspaces where it accumulates. Investigation of Aβ by laser flashphotolysis confirmed the peptide's SOD-like activity, suggesting that Aβmay be an anti-oxidant under physiological circumstances. Since H₂O₂ hasbeen shown to induce the production of Aβ, the accumulation of Aβ in ADmay reflect a response to an oxidant stress paradoxically caused by Aβexcess. This may cause and, in turn, be compounded by, damage to thebiometal homeostatic mechanisms in the brain environment.

[0165] Thus, it has recently been discovered (i) that much of the Aβaggregate in AD-affected brain is held together by zinc and copper, (ii)that Aβ peptides exhibit Fe/Cu-dependent redox activity similar to thatof SOD, (iii) that Aβ₁₋₄₂ is especially redox reactive and has theunusual property of reducing O₂ to O₂ ⁻, and (iv) that deregulation ofAβ redox reactivity causes the peptide to conveniently polymerize. Sincethese reactions must be strongly implicated in the pathogenetic eventsof AD, they offer promising targets for therapeutic drug design.

[0166] The discovery that Aβ can generate H₂O₂ and Cui, both of whichare associated with neurotoxic effects, offers an explanation for theneurotoxicity of Aβ polymers. These findings suggest that it may bepossible to lessen the neurotoxicity of Aβ by controlling factors whichalter the concentrations of Cu⁺ and ROS, including hydrogen peroxide,being generated by accumulated and soluble Aβ. It has been discoveredthat manipulation of factors such as zinc, copper, and pH can result inaltered Cu⁺ and H₂O₂ production by Aβ. Therefore, agents identified asbeing useful for the adjustment of the pH and levels of zinc and copperof the brain interstitium can be used to adjust the concentration of Cu⁺and H₂O₂, and can therefore be used to reduce the neurotoxic burden.Such agents will thus be a means of treating Alzheimer's disease.

[0167] Agents Useful in the Treatment of AD

[0168] A further aspect of the present invention is predicted in part onthe elucidation of mechanisms of neurotoxicity in the brain in ADsubjects. One mechanism involves a novel O₂ ⁻ and biometal-dependentpathway of free radical generation by Aβ peptides. The radicals of thisaspect of the present invention may comprise reactive oxygen species(ROS) such as but not limited to O₂ ⁻ and OH as well as radicalized Aβpeptides. It is proposed, according to the present invention, that byinterfering in the radical generating pathway, the neurotoxicity of theAβ peptides is reduced.

[0169] Accordingly, one aspect of the present invention contemplates amethod for treating Alzheimer's disease (AD) in a subject, said methodcomprising administering to said subject an effective amount of an agentwhich is capable of inhibiting or otherwise reducing metal-mediatedproduction of free radicals.

[0170] The preferred agents according to this aspect are metalchelators, metal complexing compounds, antioxidants and compoundscapable of reducing radical formation of Aβ peptides or mediated by Aβpeptides. Particularly preferred metal chelators and metal complexorsare capable of interacting with metals (M) having either a reducedcharge state M^(n+) or an oxidized state of M^((n+1)+). Even moreparticularly, M is Fe and/or Cu.

[0171] It is proposed that interactions of Aβ with Fe and Cu are ofsignificance to the genesis of the oxidation insults that are observedin the AD-affected brain. This is due to redox-active metal ions beingconcentrated in brain neurons and participating in the generation of ROSor other radicals by transferring electrons in their reduced state anddescribed in the following reactions:

[0172] Reduced Fe/Cu reacts with molecular oxygen to generate thesuperoxide anion.

M^(n+)+O₂→M^((n+1)+)+O₂ ⁻  Reaction (1)

[0173] The O₂ ⁻ generated undergoes dismutation to H₂O₂ either catalyzedby SOD or spontaneously.

O₂ ⁻+O₂ ⁻+2H⁺→H₂O₂₊O₂  Reaction (2)

[0174] The reaction of reduced metals with H₂O₂ generates the highlyreactive hydroxyl radical by the Fenton reaction.

M^(n+)+H₂O₂→M^((n+1)+)+OH+OH⁻  Reaction (3)

[0175] Additionally, the Haber-Weiss reaction can form OH in a reactioncatalyzed by M^((n+1)+)/M^(n+) (Miller et al., 1990).

O₂ ⁻+H₂O₂→OH+OH⁻+O₂  Reaction (4)

[0176] Still more preferably, the agent comprises one or more ofbathocuproine and/or bathophenanthroline or compounds related thereto atthe structural and/or functional levels. Reference to compounds such asbathocuproine and bathophenanthroline include functional derivatives,homologues and analogues thereof.

[0177] Accordingly, another aspect of the present invention provides amethod for treating AD in a subject said method comprising administeringto said subject Ian effective amount of an agent comprising at least onemetal chelator and/or metal complexing compound for a time and underconditions sufficient to inhibit I or otherwise reduce metal-mediatedproduction of free radicals.

[0178] In one aspect, the free radicals are reactive oxygen species suchas O₂ ⁻ or OH. In another aspect, the free radicals include forms of Aβ.However, in a broader sense, it has been found that the metal-mediatedAβ reactions in the brain of AD patients results in the generation ofreduced metals and hydrogen peroxide, as well as superoxide and hydroxylradicals. Furthermore, formation of any other radical or reactive oxygenspecies by interaction of any of these products with any other metabolicsubstrate (e.g., superoxide+nitric acid=peroxynitrite) contributes tothe pathology observed in AD and Down's syndrome patients. Cu²⁺ reactionwith Aβ generates Cu⁺, Aβ., O₂ ⁻, H₂O₂, and OH., all of which not onlydirectly damage the cells, but also react with biochemical substrateslike nitric oxide.

[0179] Yet a further aspect of the present invention is directed to amethod for treating AD in a subject, said method comprisingadministering to said subject an effective amount of an agent, saidagent comprising a metal chelator, metal complexing compound or acompound capable of interfering with metal mediated free radicalformation mediated by Aβ peptides for a time and under conditionssufficient to inhibit or otherwise reduce production of radicals.

[0180] The preferred metals according to these aspects of the presentinvention include Cu and Fe and their various oxidation states. Mostpreferred are reduced forms of copper (Cu⁺) and iron (Fe²⁺).

[0181] Another mechanism elucidated in accordance with the presentinvention concerns the formation of aggregates of Aβ, as in conditionsinvolving amyloidosis. In a preferred embodiment, the aggregates arethose of amyloid plaques occurring in the brains of AD-affectedsubjects.

[0182] The aggregates according to this aspect of the present inventionare non-fibrillary and fibrillary aggregates and are held together bythe presence of a metal such as zinc and copper. A method of treatmentinvolves resolubilizing these Aβ aggregates.

[0183] The data indicate that Zn-induced Aβ1-40 aggregation iscompletely reversible in the presence of divalent metal ion chelatingagents. This suggests that zinc binding may be a reversible, normalfunction of Aβ and implicates other neurochemical mechanisms in theformation of amyloid. A process involving irreversible Aβ aggregation,such as the polymerization of Aβ monomers, in the formation of polymericspecies of Aβ that are present in amyloid plaques is thus a moreplausible explanation for the formation of neurotoxic polymeric Aβspecies.

[0184] According to this aspect of the present invention, there isprovided a method of treating AD in a subject comprising administeringto said subject an agent capable of promoting, inducing or otherwisefacilitating resolubilization of amyloid deposits for a time and underconditions to effect said treatment.

[0185] With respect to this aspect of the present invention, it isproposed that a metal chelator or metal complexing agent beadministered. Aβ deposits which are composed of fibrillary andnon-fibrillary aggregates may be resolubilized by the metal chelating ormetal complexing agents, according to this aspect. While fibrileaggregations per se, may not be fully disassociated by administration ofsuch agents, overall deposit resolubilization approaches 70%.

[0186] In addition, the agent of this aspect of the present inventionmay comprise a metal chelator or metal complexing agent alone or incombination with another active ingredient such as but not limited torifampicin, disulfiram, indomethacin or related compounds. Preferredmetal chelators are DTPA, bathocuproine, bathophenanthroline, andpenicillamine or related compounds.

[0187] A “related” compound according to these and other aspects of thepresent invention are compounds related to the levels of structure orfunction and include derivatives, homologues and analogues thereof.

[0188] Accordingly, the present invention contemplates compositions suchas pharmaceutical compositions comprising an active agent and one ormore pharmaceutically, acceptable carriers and/or diluents. The activeagent may be a single compound such as a metal chelator or metalcomplexing agent or may be a combination of compounds such as a metalchelating or complexing compound and another compound. Preferred activeagents include, for reducing radical formation, bathocuproine and/orbathophenanthroline; and for promoting resolubilization, DTPA,bathocuproine, bathophenanthroline, and penicillamine or derivatives,homologues or analogues thereof, or any combination thereof.

[0189] Further, it has been found that the agents of the presentinvention may be administered along with the compound clioquinol.Clioquinol has been shown to be particularly effective in resolubilizingAβ aggregates in combination with other chelators. Most preferrablyclioquinol is administered in combination with bathocuproine.

[0190] It has also been found that there is a clioquinol concentration“window” within which the Aβ aggregates are dissolved. Increasing theconcentration of clioquinol above the optimal window concentration notonly is toxic to the patient but also sharply drops the dissolutioneffect of clioquinol on the Aβ amyloid. Similarly, amounts below that ofthe window are too small to result in any dissolution.

[0191] Therefore, for each given patient, the attending physician needbe mindful of the window effect and attend to varying the dosages ofclioquinol so that during the course of administration, clioquinolconcentrations would be varied frequently to randomly allow achievingthe most effective concentration for dissolving Aβ amyloid deposits inthe given patient.

[0192] It is, therefore, desired that the plasma levels of clioquinolnot be steady state, but be kept fluctuating between 0.01 μM, but notgreater than 2 μM. Since the drug is absorbed to reach peak plasmalevels within 30 minutes of oral ingestion, and since the excretion halflife is about 1-3 hours, the best way to dose the patient is with oraldoses no more often than every three hours, preferably every six hoursor eight hours, but as infrequently as once every day or once every twodays are expected to be therapeutic.

[0193] An oral dose of 200 mg/kg achieves 5 μM plasma levels in rats,and 10-30 μM in dogs. An oral dose of 500 mg/kg achieves 20-70 μM inmonkeys. The drug is freely permeable into the brain and is rapidlyexcreted.

[0194] Therefore, in humans, it is expected that a plasma level of 0.5μM would be achieved within 30 minutes of ingesting 10 mg/kg bodyweight. In a 70 kg person this is 700 mg of clioquinol. Therefore, adose of 700 mg four times a day (2800 mg/day) would be therapeutic.

[0195] However, sustained treatment with doses of clioquinol at a doseas low as 10 mg/kg/day causes the neurological side effect, subacutemyclo-optic neuritis. Therefore, dosage that high is undesirable. Thisis equivalent to 700 mg/day. The side effect is believed to be due toloss of vitamin B12. Therefore, co-therapy with vitamin B12 100 μM/dayorally or, preferably, 1000 μM/month intramuscularly, is to beadministered with clioquinol treatment to abolish this side effect.

[0196] To minimize the chances of this side effect, a lower dose ofclioquinol can also be used −100 mg, three or four times a day wouldachieve peak plasma levels of about 0.1 μM, and is likely to betherapeutic without putting the patient at risk for neurological sideeffects. Nevertheless, co-administration of Vitamin B12 should bemandatory.

[0197] For the treatment of moderately affected or severely affectedpatients, where risking the neurological side effects is less of aconcern since the quality of their life is very poor, the patient may beput on a program of treatment (after informed consent) consisting ofhigh dose clioquinol for 1 to 21 days, but preferably no more than 14days, followed by a period of low dose therapy for seven days to threemonths. A convenient schedule would be two weeks of high dose therapyfollowed by two weeks of low dose therapy, oscillating between high andlow dose periods for up to 12 months. If after 12 months the patient hasmade no clinical gains on high/low clioquinol therapy, the treatmentshould be discontinued. All regimens would be accompanied by Vitamin B12co-therapy.

[0198] Another typical case would be the treatment of a mildly affectedindividual. Such a patient would be treated with low dose clioquinol forup to 12 months. If after 6 months no clinical gains have been made, thepatient could then be placed on the high/low alternation regimen for upto another 12 months.

[0199] Particular concentrations and modes of therapy will varydepending on the particular clioquinol-containing combinationadministered.

[0200] Accordingly, the present invention contemplates compositions suchas pharmaceutical compositions comprising an active agent and one ormore pharmaceutically, acceptable carriers and/or diluents. The activeagent may be clioquinol or a combination of clioquinol and another metalchelating compound.

[0201] The pharmaceutical forms containing the active agents may beadministered in any convenient manner either orally or parenteraly suchas by intravenous, intraperitoneal, subcutaneous, rectal, implant,transdermal, slow release, intrabuccal, intracerebral or intranasaladministration. Generally, the active agents need to pass the bloodbrain barrier and may have to be chemically modified, e.g. madehydrophobic, to facilitate this or be administered directly to the brainor via other suitable routes. For injectable use, sterile aqueoussolutions (where water soluble) are generally used or alternativelysterile powders for the extemporaneous preparation of sterile injectablesolutions may be used. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and vegetable oils.The preventions of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

[0202] Sterile injectable solutions are prepared by incorporating theactive agents in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby sterilization by, for example, filtration or irradiation. In the caseof sterile powders for the preparation of sterile injectable solutions,the preferred methods of preparation are vacuum drying and thefreeze-drying technique which yield a powder of the active ingredientplus any additional desired ingredient from previously sterile-filteredsolution thereof. Preferred compositions or preparations according tothe present invention are prepared so that an injectable dosage unitcontains between about 0.25 μg and 500 mg of active compound.

[0203] When the active agents are suitably protected they may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or it may be enclosed in hard or soft shell gelatincapsule, or it may be compressed into tablets, or it may be incorporateddirectly with the food of the diet. For oral therapeutic administration,the active compound may be incorporated with excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 1% by weight of active compound.The percentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 5 to about 80% of theweight of the unit. The amount of active compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained. Preferred compositions or preparations according to thepresent invention are prepared so that an oral dosage unit form containsbetween about 1.0 μg and 2000 mg of active compound.

[0204] The tablets, troches, pills, capsules and the like may alsocontain other components such as listed hereafter: A binder such as gum,acacia, corn starch or gelatin; excipients such as dicalcium phosphate;a disintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such a sucrose, lactose or saccharin may be added or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound(s) may be incorporated intosustained-release preparations and formulations.

[0205] Pharmaceutically acceptable carriers and/or diluents include anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutical active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active ingredient, use thereof in thetherapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

[0206] It is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the novel dosage unit forms of the invention are dictated by anddirectly dependent on (a) the unique characteristics of the activematerial and the particular therapeutic effect to be achieved, and (b)the limitations inherent in the art of compounding such an activematerial for the treatment of disease in living subjects having adiseased condition in which bodily health is impaired as hereindisclosed in detail.

[0207] The principal active ingredient is compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form as hereinbeforedisclosed. A unit dosage form can, for example, contain the principalactive compound in amounts ranging from 0.5 fig to about 2000 mg.Alternatively, amounts ranging from 200 ng/kg/body weight to above 10mg/kg/body weight may be administered. The amounts may be for individualactive agents or for the combined total of active agents.

[0208] Compositions of the present invention include all compositionswherein the compounds of the present invention are contained in anamount which is effective to achieve their intended purpose. They may beadministered by any means that achieve their intended purpose. Thedosage administered will depend on the age, health, and weight of therecipient, kind of concurrent treatment, if any, frequency of thetreatment, and the nature of the effect desired. The dosage of thevarious compositions can be modified by comparing the relative in vivopotencies of the drugs and the bioavailability using no more thanroutine experimentation.

[0209] The pharmaceutical compositions of the invention may beadministered to any animal which may experience the beneficial effectsof the compounds of the invention. Foremost among such animals aremammals, e.g., humans, although the invention is not intended to be solimited.

[0210] The following examples are provided by way of illustration tofurther describe certain preferred embodiments of the invention, and arenot intended to be limiting of the present invention, unless specified.

EXAMPLES Example 1 Copper-induced, pH Dependent Aggregation of Aβ

[0211] Materials and Methods

[0212] a) Preparation of Aβ Stock

[0213] Human Aβ peptide was synthesized, purified and characterized byHPLC analysis, amino acid analysis and mass spectroscopy by W. M. KeckFoundation Biotechnology Resource Laboratory (Yale University, NewHaven, Conn.). Synthetic Aβ peptide solutions were dissolved intrifluoroethanol (30% in Milli-Q water (Millipore Corporation, Milford,Mass.)) or 20 mM HEPES (pH 8.5) at a concentration of 0.5-1.0 g/ml,centrifuged for 20 min. at 10,000 g and the supernatant (stock Aβ) usedfor subsequent aggregation assays on the day of the experiment. Theconcentration of stock Aβ was determined by UV spectroscopy at 214 nm orby Micro BCA protein assay (Pierce, Rockford, Ill.). The Micro BCA assaywas performed by adding 10 μl of stock Aβ (or bovine serum albuminstandard) to 140 μl of distilled water, and then adding an equal volumeof supernatant (150 μl) to a 96-well plate and measuring the absorbanceat 562 nm. The concentration of Aβ was determined from the BSA standardcurve. Prior to use all buffers and stock solutions of metal ions werefiltered though a 0.22 μm filter (Gelan Sciences, Ann Arbor, Mich.) toremove any particulate matter. All metal ions were the chloride salt,except lead nitrate.

[0214] b) Aggregation Assays

[0215] Aβ stock was diluted to 2.5 μM in 150 mM NaCl and 20 mM glycine(pH 3-4.5), MES (pH 5-6.2) or HEPES (pH 6.4-8.8), with or without metalions, incubated (30 min., 37° C.), centrifuged (20 min., 10000 g). Theamount of protein in the supernatant was determined by the Micro BCAprotein assay as described above.

[0216] c) Turbidometric Assays

[0217] Turbidity measurements were performed as described by Huang, X.,et al., J. Biol. Chem. 272:26464-26470 (1997), except Aβ stock wasbrought to 10 μM (300 μl) in 20 mM HEPES buffer, 150 mM NaCl (pH 6.6,6.8 or 7.4) with or without metal ions prior to incubation (30 min., 37°C.). To investigate the pH reversibility of Cu²⁺-induced Aβ aggregation,25 μM Aμ₁₋₄₀ and 25 μM Cu²⁺ were mixed in 67 mM phosphate buffer, 150 mMNaCl (pH 7.4) and turbidity measurements were taken at four 1 min.intervals. Subsequently, 20 μl aliquots of 10 mM EDTA or 10 mM Cu²⁺ wereadded into the wells alternatively, and, following a 2 min. delay, afurther four readings were taken at 1 min. intervals. After the finalEDTA addition and turbidity reading, the mixtures were incubated for anadditional 30 min. before taking final readings. To investigate thereversibility of pH mediated Cu²⁺-induced Aβ₁₋₄₀ aggregation, 10 μM Aβand 30 μM Cu²⁺ were mixed in 67 mM phosphate buffer, 150 mM NaCl (pH7.4) and an initial turbidity measurement taken. Subsequently, the pH ofthe solution was successively decreased to 6.6 and then increased backto 7.5. The pH of the reaction was monitored with a microprobe (LazarResearch Laboratories Inc., Los Angeles, Calif.) and the turbidity readat 5 min. intervals for up to 30 min. This cycle was repeated threetimes.

[0218] d) Immunofittration Detection of Low Concentrations of Aβ₁₋₄₀Aggregate

[0219] Physiological concentrations of Aβ (8 nM) were brought to 150 mMNaCl, 20 mM HEPES (pH 6.6 or 7.4), 100 nM BSA with CuCl₂ (0, 0.1, 0.2,0.5 and 2 μM) and incubated (30 min., 37° C.). The reaction mixtures(200 μl) were then placed into the 96-well Easy-Titer ELIFA system(Pierce, Rockford, Ill.) and filtered through a 0.22 μm celluloseacetate filter (MSI, Westboro, Mass.). Aggregated particles were fixedto the membrane (0.1% glutaraldehyde, 15 min.), washed thoroughly andthen probed with the anti-Aβ mAB 6E10 (Senetek, Maryland Heights,Mich.). Blots were washed and exposed to film in the presence of ECLchemiluminescence reagents (Amersham, Buckinghamshire, England).Immunoreactivity was quantified by transmitance analysis of ECL filmfrom the immunoblots.

[0220] e) Aβ Metal-capture ELISA

[0221] Aβ (1.5 ng/well) was incubated (37° C., 2 hr) in the wells of Cutcoated microtiter plates (Xenopore, Hawthorne, N.J.) with increasingconcentrations of Cu²⁺ (1-100 nM). Remaining ligand binding sites onwell surfaces were blocked with 2% gelatin in tris-buffered saline (TBS)(3 hr at 37° C.) prior to overnight incubation at room temperature withthe anti-Aβ mAb 6E10 (Senetek, Maryland Heights, Mich.). Anti-mouse IgGcoupled to horseradishperoxidase was then added to each well andincubated for 3 hr at 37° C. Bound antibodies were detected by a 30minute incubation with stable peroxidase substratebuffer/3,3′,5,5′-Tetramethyl benzidine (SPSB/TMB) buffer, followed bythe addition of 2 M sulfuric acid and measurement of the increase inabsorbance at 450 nm.

[0222] f) Extraction of Aβ from Post-mortem Brain Tissue

[0223] Identical regions of frontal cortex (0.5 g) from post-mortembrains of individuals with AD, as well as non-AD conditions, werehomogenized in TBS, pH 4.7 ±metal chelators. The homogenate wascentrifuged and samples of the soluble supernatant as well as the pelletwere extracted into SDS sample buffer and assayed for Aβ content bywestern blotting using monoclonal antibody (mAb) WO2. The data shows atypical (of n=12 comparisons) result comparing the amount of Aβextracted into the supernatant phase in AD compared to control (youngadult) samples. N,N,N′,N′-tetrakis [2-pyridyl-methyl] ethylenediamine(TPEN) (5 μM) allows the visualization of a population of pelletable Aβthat had not previously been recognized in unaffected brain samples(FIG. 8).

[0224] g) Aβ Polymerization by Copper

[0225] Cu²⁺-induced SDS-resistant oligomerization of Aβ: Aβ (2.5 μM),150 mM NaCl, 20 mM hepes (pH 6.6, 7.4, 9) with or without ZnCl₂ orCuCl₂. Following incubation (37° C.), aliquots of each reaction (2 ngpeptide) were collected at 0 d, 1 d, 3 d and 5 d and western blottedusing anti-Aβ monoclonal antibody 8E10 (FIG. 9). Migration of themolecular size markers are indicated (kDa). The dimer formed under theseconditions has been found to be SDS-resistant. Cu²⁺ (2-30 μM) inducedSDS-resistant polymerization of peptide. Co-incubation with similarconcentrations of Zn²⁺ accelerates the polymerization, but zinc alonehas no effect. The antioxidant sodium metabisulfite moderatelyattenuates the reaction, while ascorbic acid dramatically accelerates Aβpolymerization. This suggests reduction of Cu²⁺ to Cu⁺ with the lattermediating SDS-resistant polymerization of Aβ. Mannitol also abolishesthe polymerization, suggesting that the polymerization is mediated bythe generation of the hydroxyl radical by a Fenton reaction thatrecruits Cu⁺. It should be noted that other means of visualizing and/ordetermining the presence or absence of polymerization other than westernblot analysis may be used. Such other means include but are not limitedto density sedimentation by centrifugation of the samples.

[0226] Results

[0227] It has previously been reported that Zn²⁺ induces rapidprecipitation of Aβin vitro (Bush, A. I., et al., Science 265:1464(1994)). This metal has an abnormal metabolism in AD and is highlyconcentrated in brain regions where Aβ precipitates. The present dataindicate that under very slightly acidic conditions, such as in thelactic acidotic AD brain, Cu²⁺ strikingly induces the precipitation ofAβ through an unknown conformational shift. pH alone dramaticallyaffects Aβ solubility, inducing precipitation when the pH of theincubation approaches the pl of the peptide (pH 5-6). Zinc induces40-50% of the peptide to precipitate at pH>6.2, below pH 6.2 theprecipitating effects of Zn²⁺ and acid are not summative. At pH<5, Zn²⁺has little effect upon Aβ solubility. Cu²⁺ is more effective than Zn²⁺in precipitating Aβ and even induces precipitation at thephysiologically relevant pH 6-7. Copper-induced precipitation of Aβoccurs as the pH falls below 7.0, comparable with conditions of acidosis(Yates, C. M., et al., J. Neurochem. 55:1624 (1990)) in the AD brain.Investigation of the precipitating effects of a host or other metal ionsin this system indicated that metal ion precipitation of Aβ was limitedto copper and zinc, as illustrated, although Fe²⁺ possesses a partialcapacity to induce precipitation (Bush, A. I., et al., Science 268:1921(1995)).

[0228] On the basis these in vitro findings, the possibility that Aβdeposits in the AD-affected brain may be held in assembly by zinc andcopper ions was investigated. Roher and colleagues have recently shownthat much of the Aβ that deposits in AD-affected cortex can besolubilized in water (Roher, A. E, et al., J. Biol. Chem. 271:20631(1996)). Supporting the clinical relevance of in vitro findings, it hasrecently been demonstrated that metal chelators increase the amount ofAβ extracted by Roher's technique (in neutral saline buffer), and thatthe extraction of Aβ is increased as the chelator employed has a higheraffinity for zinc or copper. Hence TPEN is highly efficient inextracting Aβ, as are TETA, and bathocuproine, EGTA and EDTA are lessefficient, requiring higher concentrations 91 mm) to achieve the samelevel of recovery as say, TPEN (5μM). Zinc and copper ions (5-50 μM)added back to the extracting solution abolish the recovery of Aβ (whichis subsequently extracted by the SDS sample buffer in the pelletfraction of the centrifuged brain homogenate suspension), but Ca²⁺ andMg²⁺ added back to the chelator-mediated extracts of Aβ cannot abolishAβ resolubilization from AD-affected tissue even when these metal ionsare present in millimolar concentrations.

[0229] Importantly, atomic absorption spectrophotometry assays of themetal content of the chelator-mediated extracts confirms that Cu and Znare co-released with Aβ by the chelators, along with lowerconcentrations of Fe. These data strongly indicate that Aβ deposits(probably of the amorphous type) are held together by Cu and Zn and mayalso contain Fe. Interestingly, Aβ is not extractable from control brainwithout the use of chelators. This suggests that metal-assembled Aβdeposits may be the earliest step in the evolution of Aβ plaquepathology.

[0230] These findings propelled further inquiries into chemistry ofmetal ion-Aβ interaction. The precipitating effects upon Aβ of Zn²⁺ andCu²⁺ were found to be qualitatively different. Zn-mediated aggregationis reversible with chelation and is not associated with neurotoxicity inprimary neuronal cell cultures, whereas Cu-mediated aggregation isaccompanied by the slow formation of covalently-bonded SDS-resistantdimers and induction of neurotoxicity. These neurotoxic SDS-resistantdimers are similar to those described by Roher (Roher, A. E, et al., J.Biol. Chem. 271:20631(1996)).

[0231] To accurately quantitate the effects of different metals and pHon Aβ solubility, synthetic human Aβ₁₋₄₀ (2.5 μM) was incubated (37° C.)in the presence of metal ions at various pH for 30 min. The resultantaggregated particles were sedimented by centrifugation to permitdetermination of soluble Aβ₁₋₄₀ in the supernatant. To determine thecentrifugation time required to completely sediment the aggregatedparticles generated under these conditions, Aβ₁₋₄₀ was incubated for 30min at 37° C. with no metal, Zn²⁺ (100 μM), Cu²⁺ (100 μM) and pH (5.5).Reaction mixtures were centrifuged at 10000 g for different times, orultracentrifuged at 100000 g for 1 h. (FIG. 1). FIG. 1 shows theproportion of soluble Aβ₁₋₄₀ remaining following centrifugation ofreaction mixtures. Aβ₁₋₄₀ was incubated (30 min., 37° C.) with no metal,under acidic conditions (pH 5.5), Zn²⁺ (100 μM) or Cu²⁺ (100 μM), andcentrifuged at 10000 g for different time intervals, or at 100,000 g(ultracentrifuged) for 1 h for comparison. All data points are means±SD, n=3.

[0232] Given that conformational changes within the N-terminal domain ofAβ are induced by modulating [H⁺] (Soto, C., et al., J. Neurochem.63:1191-1198 (1994)), and that there is a metal (Zn²⁺) binding domain inthe same region, experiments were designed to determine whether therewas a synergistic effect of pH on metal ion-induced Aβ aggregation.Aβ₁₋₄₀ was incubated with different bioessential metal ions at pH 6.6,6.8 and 7.4. The results are show in FIG. 2A, where “all metals”indicates incubation with a combination containing each metal ion at thenominated concentrations, concurrently. FIG. 2A shows the proportion ofsoluble Aβ₁₋₄₀ remaining in the supernatant after incubation (30 min.,37° C.) with various metals ions at pH 6.6, 6.8 or 7.4 aftercentrifugation (10,000 g, 20 min.).

[0233] The [H⁺] chosen represented the most extreme, yet physiologicallyplausible [H⁺] that Aβ₁₋₄₀ would be likely to encounter in vivo. Theability of different bioessential metal ions to aggregate Aβ₁₋₄₀ atincreasing H⁺ concentrations fell into two groups; Mg²⁺, Ca²⁺, Al³⁺,Co²⁺, Hg²⁺, Fe³⁺, Pb²⁺ and Cu²⁺ showed increasing sensitivity to induceAβ₁₋₄₀,aggregation, while Fe²⁺, Mn²⁺, Ni²⁺, and Zn²⁺ were insensitive toalterations in [H⁺] in their ability to aggregate Aβ₁₋₄₀. Cu²⁺ and Hg²⁺induced most aggregation as the [H⁺] increased, although the [H⁺]insensitive Zn²⁺-induced aggregation produced a similar amount ofaggregation. Fe²⁺, but not Fe³⁺, also induced considerable aggregationas the [H⁺] increased, possibly reflecting increased aggregation as aresult of increased crosslinking of the peptide.

[0234] Similar results were obtained when these experiments wererepeated using turbidometry as an index of aggregation (FIG. 2B). Thedata indicate the absorbance changes between reaction mixtures with andwithout metal ions at pH 6.6, 6.8 or 7.4. Thus, Aβ₁₋₄₀ has both a pHinsensitive and a pH sensitive metal binding site. At higherconcentrations of metal ions this pattern was repeated, except Co²⁺ andAl³⁺-induced Aβ aggregation became pH insensitive, and Mn becamesensitive (FIG. 2C).

[0235] Since ⁶⁴Cu is impractically short-lived (t½=13 h), a novelmetal-capture ELISA assay was used to perform competition analysis ofAβ₁₋₄₀ binding to a microtiter plate impregnated with Cu²⁺, as describedin Materials and Methods. Results are shown in FIG. 3. All assays wereperformed in triplicate and are means ±SD, n=3. Competition analysisrevealed that Aβ₁₋₄₀ has at least one high affinity, saturable Cu²⁺binding site with a Kd=900 pM at pH 7.4 (FIG. 3). The affinity of Aβ forCu²⁺ is higher than that for Zn²⁺ (Bush, A. I., et al., J. Biol. Chem.269:12152 (1994)). Since Cu²⁺ does not decrease Zn²⁺-induced aggregation(Bush, A. I., et al., J. Biol. Chem. 269:12152 (1994)), indicating Cu²⁺does not displace bound Zn²⁺, there are likely to be two separate metalbinding sites. This is supported by the fact that there is both a pHsensitive and insensitive interaction with different metal ions.

[0236] Since the conformational state and solubility of Aβ is altered atdifferent pH (Soto, C., et al., J. Neurochem. 63:1191-1198 (1994)), theeffects of [H⁺] on Zn²⁺- and Cu⁺-induced Aβ₁₋₄₀ aggregation werestudied. Results are shown in FIGS. 4A, 4B and 4C. FIG. 4A shows theproportion of soluble Aβ₁₋₄₀ remaining in the supernatant followingincubation (30 min., 37° C.) at pH 3.0-8.8 in buffered saline ±Zn²⁺ (30μM) or Cu²⁺ (30 μM) and centrifugation (10000 g, 20 min.), expressed asa percentage of starting peptide. All data points are means ±SD, n=3.[H⁺] alone precipitates Aβ₁₋₄₀ (2.5 μM) as the solution is lowered belowpH 7.4, and dramatically once the pH falls below 6.3 (FIG. 4A). At pH5.0, 80% of the peptide is precipitated, but the peptide is notaggregated by acidic environments below pH 5, confirming and extendingearlier reports on the effect of pH on Aβ solubility (Burdick, D., J.Biol. Chem. 267:546-554 (1992)). Zn²⁺ (30 μM) induced a constant level(˜50%) of aggregation between pH 6.2-8.5, while below pH 6.0,aggregation could be explained solely by the effect of [H⁺].

[0237] In the presence of Cu²⁺ (30 μM), a decrease in pH from 8.8 to 7.4induced a marked drop in Aβ₁₋₄₀ solubility, while a slight decreasebelow pH 7.4 strikingly potentiated the effect of Cu²⁺ on the peptide'saggregation. Surprisingly, Cu²⁺ caused >85% of the available peptide toaggregate by pH 6.8, a pH which plausibly represents a mildly acidoticenvironment. Thus, conformational changes in Aβ brought about by smallincreases in [H⁺] result in the unmasking of a second metal binding sitethat leads to its rapid self-aggregation. Below pH 5.0, the ability ofboth Zn²⁺ and Cu²⁺ to aggregate Aβ was diminished, consistent with thefact that Zn binding to Aβ is abolished below pH 6.0 (Bush, A. I., etal., J. Biol. Chem. 269:12152 (1994)), probably due to protonation ofhistidine residues.

[0238] The relationship between pH and Cu²⁺ on Aβ₁₋₄₀ solubility wasthen further defined by the following experiments (FIG. 4B). Theproportion of soluble Aβ₁₋₄₀ remaining in the supernatant afterincubation (30 min., 37° C.) at pH 5.4-7.8 with different Cu²⁺concentrations (0, 5, 10, 20, 30 μM), and centrifugation (10,000 g, 20min.), was measured and expressed as a percentage of starting peptide.All data points are means ±SD, n=3. At pH 7.4, Cu²⁺-inducedAβaggregation was 50% less than that induced by Zn²⁺ over the sameconcentration range, consistent with earlier reports (Bush, A. I., etal., J. Biol. Chem. 269:12152 (1994)). There was a potentiatingrelationship between [H⁺] and [Cu²⁺] in producing Aβ aggregation; as thepH fell, less Cu²⁺ was required to induce the same level of aggregation,suggesting that [H⁺] is controlling Cu²⁺induced Aβ₁₋₄₀ aggregation.

[0239] To confirm that this reaction occurs at physiologicalconcentrations of Aβ₁₋₄₀ and Cu²⁺, a novel filtration immunodetectionsystem was employed. This technique enabled the determination of therelative amount of Aβ₁₋₄₀ aggregation in the presence of differentconcentrations of H⁺ and Cu²⁺ (FIG. 4C). The relative aggregation of nMconcentrations of Aβ₁₋₄₀ at pH 7.4 and pH 6.6 in the presence ofdifferent Cu²⁺ concentrations (0, 0.1, 0.2, 0.5 μM) were determined bythis method. Data represent mean reflectance values of immunoblotdensitometry expressed as a ratio of the signal obtained when thepeptide is treated in the absence of Cu²⁺. All data points are means±SD, n=2.

[0240] This sensitive technique confirmed that physiologicalconcentrations of Aβ₁₋₄₀ are aggregated under mildly acidic conditionsand that aggregation was greatly enhanced by the presence of Cu²⁺ atconcentrations as low as 200 μM. Furthermore, as previously observed athigher Aβ₁₋₄₀ concentrations, a decrease in pH from 7.4 to 6.6potentiated the effect of Cu²⁺ on aggregation of physiologicalconcentrations of Aβ₁₋₄₀. Thus, Aβ₁₋₄₀ aggregation is concentrationindependent down to 8 nM where Cu²⁺ is available.

[0241] It has recently been shown that Zn²⁺ mediated Aβ₁₋₄₀ aggregationis reversible whereas Aβ₁₋₄₀ aggregation induced by pH 5.5 wasirreversible. Therefore, experiments were performed to determine whetherCu²⁺/pH-mediated Aβ₁₋₄₀ aggregation was reversible. Cu²⁺-induced Aβ₁₋₄₀aggregation at pH 7.4 was reversible following EDTA chelation, althoughfor each new aggregation cycle, complete resolubilization of theaggregates required a longer incubation. This result suggested that amore complex aggregate is formed during each subsequent aggregationcycle, preventing the chelator access to remove Cu²⁺ from the peptide.This is supported by the fact that complete resolubilization occurs withtime, and indicates that the peptide is not adopting a structuralconformation that is insensitive to Cu²⁺-inducedaggregation/EDTA-resolubilization.

[0242] The reversibility of pH potentiated Cu²⁺-induced Aβ₁₋₄₀aggregation was studied by turbidometry between pH 7.5 to 6.6,representing H⁺ concentration extremes that might be found in vivo(FIGS. 5A and 5B). Unlike the irreversible aggregation of Aβ₁₋₄₀observed at pH 5.5. Cu²⁺-induced Aβ1-40 aggregation was fully reversibleas the pH oscillated between pH 7.4 and 6.6. FIG. 5A shows theturbidometric analysis of Cu²⁺-induced Aβ₁₋₄₀ aggregation at pH 7.4reversed by successive cycles of chelator (EDTA), as indicated. FIG. 5Bshows turbidometric analysis of the reversibility of Cu²⁺-induced Aβ₁₋₄₀as the pH cycles between 7.4 and 6.6. Thus, subtle conformationalchanges within the peptide induced by changing [H⁺] within a narrow pHwindow, that corresponds to physiologically plausible [H⁺], allows theaggregation or resolubilization of the peptide in the presence of Cu²⁺.

[0243] Discussion

[0244] These results suggest that subtle conformational changes in Aβinduced by [H⁺] promote the interaction of Aβ₁₋₄₀ with metal ions, inparticular Cu²⁺and Hg²⁺ allowing self-aggregate or resolubilizedepending on the [H⁺] (FIGS. 2A-2C, 4A-4C). A decrease in pH below 7.0increases the P-sheet conformation (Soto, C., et al., J. Neurochem.63:1191-1198 (1994)), and this may allow the binding of Cu²⁺ to solubleAβ that could further alter the conformation of the peptide allowing forself aggregation, or simply help coordinate adjacent Aβ molecules in theassembly of the peptides into aggregates. Conversely, increasing pHabove 7.0 promotes the α-helical conformation (Soto, C., et al., J.Neurochem. 63:1191-1198 (1994)), which may alter the conformationalstate of the dimeric aggregated peptide, releasing Cu and therebydestabilizing the aggregate with the resultant release of Aβ intosolution. Thus, in the presence of Cu²⁺, Aβ₁₋₄₀ oscillates between anaggregated and soluble state dependent upon the [H⁺].

[0245] Aβ₁₋₄₀ aggregation by Co²⁺, like Zn²⁺, was pH insensitive and permole induced a similar level of aggregation. Unlike Zn²⁺, Aβ₁₋₄₀ bindingof Co²⁺ may be employed for the structural determination of the pHinsensitive binding site given its nuclear magnetic capabilities (SeeFIG. 2C).

[0246] The biphasic relationship of Aβ solubility with pH mirrors theconformational changes previously observed by CD spectra within theN-terminal fragment (residues 1-28) of AD (reviewed in (Soto, C., etal., J. Neurochem. 63:1191-1198 (1994)); α-helical between pH 1-4and >7, but β-sheet between pH 4-7. The irreversible aggregates of Aβformed at pH 5.5 supports the hypothesis that the 1-sheet conformationis a pathway for Aβ aggregation into amyloid fibrils. Since aggregatesproduced by Zn²⁺ and Cu²⁺ under mildly acidic conditions (FIGS. 5A and5B) are chelator/pH reversible, their conformation may be the higherenergy α-helical conformation.

[0247] These results now indicate that there are three physiologicallyplausible conditions which could aggregate Aβ: pH (FIGS. 1, 4A-4C;Fraser, P. E., et al., Biophys. J. 60:1190-1201 (1991); Barrow, C. J.and Zagorski, M. G., Science 253:179-182 (1991); Burdick, D., J. Biol.Chem. 267:546-554 (1992); Barrow, C. J., et al., J. Mol. Biol.225:1075-1093 (1992); Zagorski, M. G. and Barrow, C. J., Biochemistry31:5621-5631 (1992); Kirshenbaum, K. and Daggett, V., Biochemistry34:7629-7639 (1995); Wood, S. J., et al., J. Mol. Biol. 256:870-877(1996), [Zn²⁺] (FIGS. 1, 2A and 2B, 4A-4C; Bush, A. I., et al., J. Biol.Chem. 269:12152 (1994); Bush, A. I., et al., Science 265:1464 (1994);Bush, A. I., et al., Science 268:1921 (1995); Wood, S. J., et al., J.Mol. Biol. 256:870-877 (1996)) and under mildly acidic conditions,[Cu²⁺] (FIGS. 2A, 4A-4C, 5B). Interestingly, changes in metal ionconcentrations and pH are common features of the inflammatory responseto injury. Therefore, the binding of Cu²⁺ and Zn²⁺ to Aβ may be ofparticular importance during inflammatory processes, since local sitesof inflammation can become acidic (Trehauf, P. S. & McCarty, D. J.,Arthr. Rheum. 14:475-484 (1971); Menkin, V., Am. J. Pathol. 10:193-210(1934)) and both Zn²⁺ and Cu²⁺ are rapidly mobilized in response toinflammation (Lindeman, R. D., et al., J. Lab. Clin. Med. 81:194-204(1973); Terhune, M. W. & Sandstead, H. H., Science 177:68-69 (1972);Hsu, J. M., et al., J. Nutrition 99:425-432 (1969); Haley, J. V., J.Surg. Res. 27:168-174 (1979); Milaninio, R., et al., Advances inInflammation Research 1:281-291 (1979); Frieden, E., in InflammatoryDiseases and Copper, Sorenson, J. R. J., ed, Humana Press, New Jersey(1980), pp. 159-169).

[0248] Serum copper levels increase during inflammation, associated withincreases in ceruloplasmin, a Cu²⁺ transporting protein that may donateCu²⁺ to enzymes active in processes of basic metabolism and woundhealing such as cytochrome oxidase and lysyl oxidase (Giampaolo, V., etal., in Inflammatory Diseases and Copper, Sorenson, J. R. J., ed, HumanaPress, New Jersey (1980), pp.329-345; Peacock, E. E. and vanWinkle, W.,in Wound Repair, W. B. Saunders Co., Philadelphia, pp. 145-155) (1976)).Since the release of Cu²⁺ from ceruloplasmin is greatly facilitated byacidic environments where the cupric ion is reduced to its cuprous form(Owen, C. A., Jr., Proc. Soc. Exp. Biol. Med. 149:681-682 (1975)),periods of mild acidosis may promote an environment of increased freeCu²⁺. Similarily, aggregation of another amyloid protein, the acutephase reactant serum amyloid P component (SAP) to the cell wallpolysaccharide, zymosan, has been observed with Cu²⁺ at acidic pH(Potempa, L. A., et al., Journal of Biological Chemistry 260:12142-12147(1985)). Thus, exchange of Cu²⁺ to Aβ₁₋₄₀ during times of decreased pHmay provide a mechanism for altering the biochemical reactivity of theprotein required by the cell under mildly acidic conditions. Such afunction may involve alterations in the aggregation/adhesive properties(FIGS. 1-5B) or oxidative functions of Aβ at local sites ofinflammation.

[0249] While the pathogenic nature of Aβ₄₂ in AD is well described(Maury, C.P.J., Lab. Investig. 72:4-16 (1995); Multhaup, G., et al.,Nature 325:733-736 (1987)), the function of the smaller Aβ₁₋₄₀ remainsunclear. The present data suggest that Cu²⁺-binding and aggregation ofAβ will occur when the pH of the microenvironment rises. This conclusioncan be based on the finding that the reaction is [H⁺] and [Cu²⁺]dependent and reversible within a narrow, physiologically plausible, pHwindow. This is further supported by the specificity and high affinityof Cu²⁺ binding under mildly acidic conditions compared to the constantZn²⁺-induced aggregation (and binding) of Aβ₁₋₄₀ over a wide pH range(6.2-8.5). The brain contains high levels of both Zn²⁺ (˜150 μM;Frederickson, C. J. International Review of Neurobiology 31:145-237(1989)) and Cu²⁺ (˜100 μM; Warren, P. J., et al., Brain 83:709-717(1960); Owen, C. A., Physiological Aspects of Copper, NoyesPublications, Park Ridge, N.J. (1982), pp160-191). Intracellularconcentrations are approximately 1000 and 100 fold higher thanextracellular concentrations. This large gradient between intracellularand extracellular compartments suggests a highly energy dependentmechanism is required in order to sequester these metals within neurons.Therefore, any alterations in energy metabolism, or injury, may affectthe reuptake of these metal ions and promote their release into theextracellular space, and together with the synergistic affects ofdecreased pH (see above) induce membrane bound Aβ₁₋₄₀ to aggregate.Since increased concentrations of Zn²⁺ and Cu²⁺, and decreased pH, arecommon features of all forms of cellular insult, the initiation ofAβ₁₋₄₀ function likely occurs in a coordinated fashion to alter adhesiveand/or oxidative properties of this membrane protein essential formaintaining cell integrity and viability. That Aβ₁₋₄₀ has such a highaffinity for these metal ions, indicates a protein that has evolved torespond to slight changes in the concentration of extracellular metalions. This is supported by the fact that aggregation in the presence ofCu is approx. 30% at pH 7.1, the pH of the brain (Yates, C. M., et al.,J. Neurochem. 55:1624-1630 (1990)), but 85% at pH 6.8. Taken together,our present results indicate that Aβ₁₋₄₀ may have evolved to respond tobiochemical changes associated with neuronal damage as part of thelocally mediated response to inflammation or cell injury. Thus, it ispossible that Cu²⁺ mediated Aβ₁₋₄₀ binding and aggregation might be apurposive cellular response to an environment of mild acidosis.

[0250] The deposition of amyloid systemically is usually associated withan inflammatory response (Pepys, M. B. & Baltz, M. L., Adv. Immunol.34:141-212 (1983); Cohen, A. S., in Arthritis and Allied Conditions, D.J. McCarty, ed., Lea and Febiger, Philadelphia, pp. 1273-1293 (1989);Kisilevsky, R., Lab. Investig. 49:381-390 (1983)). For example, serumamyloid A, one of the major acute phase reactant proteins that iselevated during inflammation, is the precursor of amyloid A protein thatis deposited in various tissues during chronic inflammation, leading tosecondary amyloidosis (Gorevic, P. D., et al., Ann. NY Acad. Sci.380:393 (1982)). An involvement of inflammatory mechanisms has beensuggested as contributing to plaque formation in AD (Kisilevsky, R.,Mol. Neurobiol 49:65-66 (1994)). Acute-phase proteins such as alpha1-antichymotrypsin and c-reactive protein, elements of the complementsystem and activated microglial and astroglial cells are consistentlyfound in AD brains.

[0251] The rapid appearance, within days of Aβ deposits and APPimmunoreactivity following head injury (Roberts, G. W., et al., Lancet.338:1422-1423 (1991); Pierce, J. E. S., et al., Journal of Neuroscience16:1083-1090 (1996)), rather than the more gradual accumulation of Aβinto more dense core amyloid plaques over months or years in AD may becompatible with the release of Zn²⁺, Cu²⁺ and mild acidosis in this timeframe. Thus, pH/metal ion mediated aggregation may form the basis forthe amorphous Aβ deposits observed in the aging brain and following headinjury, allowing the maintenance of endothelial and neuronal integritywhile limiting the oxidative stress associated with injury that may leadto a diminishment of structural function.

[0252] Since decreased cerebral pH is a complication of aging (Yates, C.M., et al., J. Neurochem. 55:1624-1630 (1990)), these data indicate thatCu and Zn mediated Aβ aggregation may be a normal cellular response toan environment of mild acidosis. However, prolonged exposure of Aβ to anenvironment of lowered cerebral pH may promote increased concentrationsof free metal ions and reactive oxygen species, and the inappropriateinteraction of Aβ₁₋₄₂ over time promoting the formation of irreversibleAβ oligomers and it's subsequent deposition as amyloid in AD. Thereversibility of this pH mediated Cu²⁺ aggregation does however presentthe potential for therapeutic intervention. Thus, cerebralalkalinization may be explored as a therapeutic modality for thereversibility of amyloid deposition in vivo.

Example 2

[0253] Free Radical Formation and SOD-like Activity of Alzheimer's AβPeptides

[0254] Materials and Methods

[0255] a) Determination of Cu⁺ and Fe²⁺

[0256] This method is modified from a protocol assaying serum copper andiron (Landers, J. W., et al., Amer. Clin. Path. 29:590 (1958)). It isbased on the fact that there are optimal visible absorption wavelengthsof 483 nm and 535 nm for Cu⁺ complexed with bathocuproinedisulfonic (BC)anion and Fe²⁺ coordinated by bathophenanthrolinedisulfonic (BP) anion,respectively.

[0257] Determination of molar absorption of these two complexes wasaccomplished essentially as follows. An aliquot of 500 μl of eachcomplex (500 μM, in PBS pH 7.4, with ligands in excess) was pipettedinto 1 cm-pathlength quartz cuvette, and their absorbances weremeasured. Their molar absorbancy was determined based on Beer-Lambert'sLaw. Cu⁺-BC has a molar absorbancy of 2762 M⁻¹ cm⁻¹, while Fe²⁺-BP has amolar absorbancy of 7124 M⁻¹ cm⁻¹.

[0258] Determination of the equivalent vertical pathlength for Cu⁺-BCand Fe²⁺-BP in a 96-well plate was carried out essentially as follows.Absorbances of the two complexes with a 500 μM, 100 μM, 50 μM, and 10 μMconcentration of relevant metal ions (Cu⁺, Fe²⁺) were determined both by96-well plate readers (300 μL) and UV-vis spectrometer (500 μL), withPBS, pH 7.4, as the control blank. The resulting absorbancies from theplate reader regress against absorbancies by a UV-vis spectrometer. Theslope k from the linear regression line is equivalent to the verticalpathlength if the measurement is carried out on a plate. The resultsare: k(cm) r² Cu⁺—BC 1.049 0.998 Fe²⁺—BP 0.856 0.999

[0259] With molar absorbancy and equivalent vertical pathlength in hand,the concentrations (μM) of Cu⁺ or Fe²⁺ can be deduced based onBeer-Lambert's Law, using proper buffers as controls.${{for}\quad {{Fe}^{2 +}:{\left\lbrack {Fe}^{2 +} \right\rbrack ({\mu M})}}} = {\frac{\Delta \quad {A\left( {535{nm}} \right)}}{\left( {7124 \times 0.856} \right)} \times 10^{6}}$${{for}\quad {{Cu}^{+}:{\left\lbrack {Cu}^{+} \right\rbrack ({\mu M})}}} = {\frac{\Delta \quad {A\left( {483{nm}} \right)}}{\left( {2762 \times 1.049} \right)} \times 10^{6}}$

[0260] where ΔA is absorbancy difference between sample and controlblank.

[0261] b) Determination of H₂O₂

[0262] This method is modified from a H₂O₂ assay reported recently (Han,J. C., et al., Anal. Biochem. 234:107 (1996)). The advantages of thismodified H₂O₂ assay on 96-well plate include high throughput, excellentsensitivity (˜1 μM), and the elimination of the need for a standardcurve of H₂O₂, which is problematic due to the labile chemical propertyof H₂O₂.

[0263] Aβ peptides were co-incubated with a H₂O₂-trapping reagent(Tris(2-carboxyethyl)-phosphine hydrochloride, TCEP, 100 μM) in PBS (pH7.4 or 7.0) at 37° C. for 30 mins. Then 5,5′-dithio-bis(2-nitrobenzoicacid) (DBTNB, 100 μM) was added to react with remaining TCEP. Theproduct of this reaction has a characteristic absorbance maximum of 412nm [18]. The assay was adapted to a 96-well format using a standardabsorbance range (see FIG. 11).

[0264] The chemical scheme for this novel method is:

[0265] TCEP.HCl was synthesized by hydrolyzing tris (2-cyno-ethyl)phosphine (purchased from Johnson-Mathey (Waydhill, Mass.)), inrefluxing aqueous HCl (Burns, J. A. et al., J. Org. Chem. 56:2648(1991)) as shown below.

[0266] In order to carry out the above-described assay in a 96-wellplate, it was necessary to calculate the equivalent vertical pathlengthof 2-nitro-5-thiobenzoic acid (TMB) in a 96-well plate. Thisdetermination was carried out essentially as described for Cu⁺-BC andFe²⁺-BP in Example 5. The resulting absorbancies from the plate readerregress against absorbancies by a UV-vis spectrometer. The slope k fromthe linear regression line is equivalent to the vertical pathlength ifthe measurement is carried out on a plate. The results are: k r² 0.8751.00

[0267] The concentration of H₂O₂ can then be deduced from the differencein absorbance between the sample and the control (sample plus 1000 U/μlcatalase)${\left\lbrack {H_{2}O_{2}} \right\rbrack ({\mu M})} = \frac{\Delta \quad {A\left( {412{nm}} \right)}}{\left( {2 \times 0.875 \times 14150} \right)}$

[0268] c) Determination of OH.

[0269] Determination of OH. was performed as described in Gufferidge etal. Biochim. Biophys. Acta 759: 38-41(1983).

[0270] d) Cu⁺ Generation by Aβ: Influence of Zn²⁺ and pH

[0271] Aβ (10 μM in PBS, pH 7.4 or 6.8, as shown) was incubated for 30minutes (37° C.) in the presence of Cu²⁺10 μM ±Zn²⁺ 10 μM). Cu³⁰ levels(n=3, ±SD) were assayed against a standard curve. These data indicatethat the presence of Zn²⁺ can mediate the reduction of Cu²⁺ in a mildlyacidic environment. The effects of zinc upon these reactions arestrongly in evidence but complex. Since the presence of 10 μM zinc willprecipitate the peptide, it is clear that the peptide possesses redoxactivity even when it is not in the soluble phase, suggesting thatcortical Aβ deposits will not be inert in terms of generating thesehighly reactive products. Cerebral zinc metabolism is deregulated in AD,and therefore levels of interstitial zinc may play an important role inadjusting the Cu⁺ and H₂O₂ production generated by Aβ. The rat homologueof Aβ₁₋₄₀ does not manifest the redox reactivity of the humanequivalent. Insulin, a histidine-containing peptide that can bind copperand zinc, exhibits no Cu²⁺ reduction.

[0272] e) Hydrogen Peroxide Production by Aβ Species

[0273] Aβ₁₋₄₂ (10 μM) was incubated for 1 hr at 37° C., pH 7.4 inambient air (first bar), continuous argon purging (Ar), continuousoxygen enrichment (O₂) at pH 7.0 (7.0), or in the presence of the ironchelator desferioxamine (220 μM; DFO). Variant Aβ species (10 μM) weretested: Aβ₁₋₄₀ (Aβ₁₋₄₀), rat Aβ₁₋₄₀ (rAβ₁₋₄₀), and scrambled Aβ₁₋₄₀(sAβ₁₋₄₀) were incubated for 1 hr at 37° C., pH 7.4 in ambient air.Values (mean ±SD, n=3) represent triplicate samples minus values derivedfrom control samples run under identical conditions in the presence ofcatalase (10 U/ml). The details of the experiment are as follows: Aβpeptides were co-incubated with a H₂O₂-trapping reagent(Tris(2-carboxyethyl)-phosphine hydrochloride, TCEP, 100 μM) in PBS (pH7.4 or 7.0) at 37° C. for 30 mins. Then 5,5′-dithio-bis(2-nitrobenzoicacid) (DTNB, 100 μM) was added to react with remaining TCEP, the producthas a characteristic absorbance maximum of 412 nm. The assay was adaptedto a 96-well format using a standard absorbance range.

[0274] Results and Discussion

[0275] Aβ exhibits metal-dependent and independent redox activity

[0276] Because Aβ was observed to be covalently linked by Cu, theability of the peptide to reduce metals and generate hydroxyl radicalswas studied. The bathocuproine and bathophenanthroline reduced metalassay technique employed by Multhaup et al. was used in order todetermine that APP itself possesses a Cu²⁺ reducing site on itsectodomain (Multhaup, G., et al., Science 271:1406 (1996)). It has beendiscovered that Aβ possesses a striking ability to reduce both Fe³⁺ toFe²⁺, and Cu²⁺ to Cu⁺, modulated by Zn²⁺ and pH (6.6-7.4) (See FIG. 10).It is of great interest that the relative redox activity of the peptidesstudied correlates so well with their relative pathogenicity vizAβ₁₋₄₂>Aβ₁₋₄₀>ratAβ in all redox assays studied. Since one of thecaveats in using the reduced metals assay is that the detection agentscan exaggerate the oxidation potential of Cu²⁺ or Fe (III), other redoxproducts were explored by assays where no metal ion indicators werenecessary. It was discovered that hydrogen peroxide was rapidly formedby Aβspecies (FIG. 11). Thus, Aβ produces both H₂O₂ and reduced metalswhilst also binding zinc. Structurally, this is difficult to envisagefor a small peptide, but we have recently shown that Aβ is dimeric inphysiological buffers. Since H₂O₂ and reduced metal species are producedin the same vicinity, these reaction products are liable to produce thehighly toxic hydroxyl radical by Fenton chemistry, and the formation ofhydroxyl radicals from these peptides has now been shown with thethiobarbituric acid assay. The formation of hydroxyl radicals correlateswith the covalent polymerization of the peptide (FIG. 9) and can beblocked by hydroxyl scavengers. Thus the concentrations of Fe, Cu, Zn &H⁺ in the brain interstitial milieu could be important in facilitatingprecipitation and neurotoxicity for Aβ by direct (dimer formation) andindirect (Fe²⁺/Cu⁺ and H₂O₂ formation) mechanisms.

[0277] H₂O₂ production by Aβ explains the mechanism by which H₂O₂ hasbeen described to mediate neurotoxicity (Behl, C. et al., Cell 77:827(1994)), previously thought to be the product of cellular overproductionalone. Interestingly, the scrambled Aβ peptide (same size and residuecontent as FIG. 6) produces appreciable H₂O₂ but no hydroxyl radicals.This is because the scrambled Aβ peptide is unable to reduce metal ions.This leads to the conclusion that what makes Aβ such a potent neurotoxinis its capacity to produce both reduced metals and H₂O₂ at the sametime. This “double whammy” can then produce hydroxyl radicals by theFenton reaction, especially if the H₂O₂ is not rapidly removed from thevicinity of the peptide. Catalase and glutathione peroxidase are theprincipal means of catabolizing H₂O₂, and their levels are low in thebrain, especially in AD, perhaps explaining the propensity of Aβ toaccumulate in brain tissue.

[0278]FIG. 11 shows that the production of H₂O₂ is oxygen dependent, andfurther investigation has indicated that Aβ can spontaneously producethe superoxide radical (O₂) in the absence of metal ions. This propertyof Aβ is particularly exaggerated in the case of Aβ₁₋₄₂, probablyexplaining why this peptide is more neurotoxic and more enriched thanAβ₁₋₄₀ in amyloid. O₂ generation will be subject to spontaneousdismutation to generate H₂O₂, however, this is a relatively slowreaction, although it may account for the majority of the H₂O₂ detectedin our Aβ assays. O₂ is reactive, and the function of superoxidedismutase (SOD) is to accelerate the dismutation to produce H₂O₂ whichis then catabolized by catalase and peroxidases into oxygen and water.The most abundant form of SOD is Cu/Zn SOD, mutations of which causeanother neurodegenerative disease, amyotrophic lateral sclerosis (Rosen,D., et al., Nature 364:362 (1993)). Since Aβ, like Cu/Zn SOD, is adimeric protein that binds Cu and Zn and reduces Cu²⁺ and Fe³⁺, westudied the O₂ dismutation behavior of Aβ in the sec time-scale usinglaser pulse photolysis. These experiments have shown that Aβ exhibitsFe/Cu-dependent SOD-like activity with rate constants of dismutation at≈10⁸ M⁻¹ sec⁻¹, which are strikingly similar to SOD. Hence, Aβ appearsto be a good candidate to possess the same function as SOD, andtherefore may function as an antioxidant. This may explain why oxidativestresses cause it to be released by cells (Frederikse, P. H., et al.,Journal of Biological Chemistry 271: 10169 (1996)). However, if Aβ₁₋₄₂is involved in the reaction to oxidative stress, or if the H₂O₂clearance is compromised at the cellular level, Aβ will accumulate,recruiting more O₂ and producing more O₂ leading to a vicious cycle andlocalizing tissue peroxidation damage and protein cross-linking.

Example 3 Therapeutic Agents for Inhibition of Metal-mediated Productionof Reactive Oxygen Species

[0279] Materials and Methods

[0280] a) Synthesis of Peptides

[0281] Synthetic Aβ peptides Aβ₁₋₄₀ and Aβ₁₋₄₂ were synthesized by theW. Keck Laboratory, Yale, Conn. In order to verify the reproducibilityof the data obtained with these peptides, confirmatory data wereobtained by reproducing experiments with these Aβ peptides synthesizedand obtained from other sources: Glabe laboratory, University ofCalifornia, Irvine, Calif., Multhaup Laboratory, University ofHeidelberg, U.S. Peptides, Bachem, and Sigma. Rat Aβ was synthesized andcharacterized by the Multhaup Laboratory, University of Heidelberg.Aβ₁₋₂₈ was purchased from U.S. Peptides, Bachem, and Sigma. Aβ peptidestock solutions were prepared in chelex-100 resin (BioRad) treated waterand quantified.

[0282] b) Metal Reduction Assay

[0283] The metal reduction assay was performed using a 96-wellmicrotiter plate (Costar) based upon a modification of establishedprotocols (Landers, J. W., et al., Amer. Clin. Path. 29:590 (1958);Landers, J. W., et al., Clinica Chimica Acta 3:329 (1958)). Polypeptides(10 μM) or Vitamin C (100 μM), metal ions (10 μM, Fe(NO₃)₃ or Cu(NO₃)₂),and reduced metal ion indicators, bathophenanthrolinedisulfonic acid(BP, for Fe²⁺, Sigma, 200 μM) or bathocuproinedisulfonic acid (BC, forCu⁺, Sigma, 200 μM), were coincubated in phosphate buffered saline(PBS), pH 7.4, for 1 hr at 37° C. The metal ion solutions were preparedby direct dilution in the buffer from their aqueous stocks purchasedfrom National Institute of Standards and Technology (NIST). Absorbanceswere then measured at 536 mn (Fe²⁺-BP complex) and 483 nm (Cu⁺-BCcomplex), respectively, using a 96-well plate reader (SPECTRAmax 250,Molecular Devices, CA). In control samples, both metal ion and indicatorwere present to determine the background buffer signal. As a furthercontrol, both metal ion and peptide were present in the absence ofindicator to estimate the contribution of light scattering due toturbidity to the absorbance reading at these wavelengths. The netabsorbances (ΔA) at 536 nm or 483 nm were obtained by deducting theabsorbances from these controls from the absorbances generated by thepeptide and metal in the presence of the respective indicator.

[0284] The concentrations of reduced metal ions (Fe²⁺ or Cu⁺) werequantified based on the formula: Fe²⁺ or Cu⁺ (˜M)=A*10⁶/(L*M), where Lis the measured equivalent vertical pathlength for a well of 300 μLvolume as described in the instrument's specifications manual (0.856 cmfor Fe²⁺; 1.049 cm for Cu⁺); M is the known molecular absorbance (M⁻¹cm⁻¹) which is 7124 (for Fe²⁺-BP complex) or 2762 (for Cu⁺-BC complex).

[0285] c) H₂O₂ Assay

[0286] The H₂O₂ assay was performed in a UV-transparent 96-wellmicrotiter plate (Molecular Devices, CA), according to a modification ofan existing protocol (Han, J. C., et al., Anal. Biochem. 234:107 (1996);Han et al., Anal. Biochem. 220: 5-10 (1994)). Polypeptides (10 μM) orVitamin C (10 μM), Fe³⁺ or Cu²⁺ (1 μM) and a H₂O₂ trappingagent-Tris(2-Carboxyethyl)Phosphine Hydrochloride (TCEP, Pierce, 50μM)-were co-incubated in PBS buffer (300 μL), pH 7.4, for 1 hour at 37°C. Under identical conditions, catalase (Sigma, 100 U/mL) wassubstituted for the polypeptides, to serve as a control signalrepresenting 0 μM H₂O₂. Following incubation, the unreacted TCEP wasdetected by 5,5-Dithio-bis(2-Nitrobenzoic acid) (DTNB, Sigma, 50 KLM)which generates 2 moles of the coloured product. The reactions are:

TCEP+H₂O₂→TCEP=O+H₂O,

[0287] then the remaining TCEP is reacted with DTNB:

TCEP+DTNB+H₂O→TCEP=O+2NTB (2-nitro-5-thiobenzoate).

[0288] The amount of H₂O₂ produced was quantified based on the formula:H₂O₂ (μM)=hA*106/(2*L*M), where hA is the absolute absorbance differencebetween a sample and catalase-only control at 412 nm wavelength; L=0.875cm, the equivalent vertical pathlength obtained from the platereadermanufacturer's specifications; M is the molecular absorbance for NTB(14150 M⁻¹ cm⁻¹ at 412 nm).

[0289] TCEP is a strong reducing agent, and, hence, will artifactuallyreact with polypeptides that contain disulfide bonds. This wasdetermined not to be a source of artifact for the measurement of H₂O₂generation from Aβ, which does not possess a disulfide bond.

[0290] d) Estimation of O₂

[0291] The spectrophotometric absorption peak for O₂ is 250 nm where itsextinction coefficient is much greater than that of H₂O₂ (Bielski etal., Philos Trans R Soc Lond B Biol Sci. 311: 473-482 (1985)). Theproduction of O₂ ⁻ was estimated by measuring the spectrophotometricabsorption of polypeptides (10 μM, 300 μL) after incubation for one hourin PBS, pH 7.4, at 37° C., using a 96-well plate reader. Thecorresponding blank was the signal from PBS alone. An absolute baselinefor the signal generated by the peptide was not achievable in this assaysince the absorption peak for tyrosine (residue 10 of Aβ) is close (254nm) to the absorption peak for O₂ ⁻. However, attenuation of theabsorbance by co-incubation with superoxide dismutase (100 U/mL)indicated that the majority of the absorbance signal was due to thepresence of O₂ ⁻.

[0292] e) Thiobarbituric Acid Reaction Substance (TBARS) Assay—OH.

[0293] The Thiobarbituric Acid-Reactive Substance (TBARS) assay forincubation mixtures with Fe³⁺ or Cu²⁺ was performed in a 96-wellmicrotiter format modified from established protocols (Gutteridge et al.Biochim. Biophys. Acta 759: 38-41 (1983)). Aβ peptide species (10 μM) orVitamin C (100 μM), were incubated with Fe³⁺ or Cu²⁺ (1 μM) anddeoxyribose (7.5 mM, Sigma) in PBS, pH 7.4. Following incubation (37°C., 1 hour), glacial (17 M) acetic acid and 2-thiobarburic acid (1%, w/vin 0.05 M NaOH, Sigma) were added and heated (100° C., 10 min). Thefinal mixtures were placed on ice for 1-3 minutes before absorbances at532 nm were measured. The net absorbance change for each sample wereobtained by deducting the absorbance from a control sample consisting ofidentical chemical components except for the Vitamin C or Aβ peptides.

[0294] Results and Discussion

[0295] Oxygen radical involvement in human aging, the predominant riskfactor for Alzheimer's disease (AD), was first proposed by Harman in1956 (Harman, D., J. Gerontol. 11:298 (1956)) and increasing evidencehas implicated oxidative stress in the pathogenesis of AD. Apart frommetabolic signs of oxidative stress in AD-affected neocortex such asincreased glucose-6-phosphate dehydrogenase activity (Martins, R. N., etal., J. Neurochem. 46:1042-1045 (1986)) and increased heme oxygenase-1levels (Smith, M. A., et al., Am. J. Pathol. 145:42 (1994)), there arealso numerous signs of oxygen radical-mediated chemical attack such asincreased protein and free carbonyls (Smith, C. D., et al., Proc. Natl.Acad. Sci. USA 88:10540 (1991); Hensley, K., et al., J. Neurochem.65:2146 (1995); Smith, M. A., et al., Nature 382:120 (1996)), lipidperoxidation adducts (Palmer, A. M. & Burns, M. A., Brain Res. 645:338(1994); Sayre, L. M. et al., J. Neurochem. 68:2092 (1997)),peroxynitrite-mediated protein nitration (Good, P. F., et al., Am. J.Pathol. 149:21 (1996)); Smith, M. A., et al., Proc. Natl. Acad. Sci. USA94:9866 (1997)), and mitochondrial and nuclear DNA oxidation adducts(Mecocci, P., et al., Ann. Neurol., 34:609-616 (1993); Mecocci, P., etal., Ann. Neurol., 36:747-751 (1994)). Recently, treatment ofindividuals with the antioxidant vitamin E has been reported to delaythe progression of clinical AD (Sano, M. et al., N. Engl. J. Med.336:1216 (1997)).

[0296] A relationship seems likely to exist between the signs ofoxidative stress and the characteristic Aβ collections (Glenner, G. G. &Wong, C., Biochem. Biophys. Res. Commun. 120:885 (1984)) found in thecortical interstitium and cerebrovascular intima media in AD. The brainregional variation of oxidation biomarkers corresponds with amyloidplaque density (Hensley, K., et al., J. Neurochem. 65:2146 (1995)).Indeed, neurons cultured from subjects with Down's syndrome, a conditioncomplicated by the invariable premature deposition of cerebral Aβ(Rumble, B., et al., N. Engl. J. Med. 320:1446 (1989)) and theoverexpression of soluble Aβ1-42 in early life (Teller, J. K., et al.,Nature Medicine 2:93 (1996)), exhibit lipid peroxidation and apoptoticcell death caused by increased generation of hydrogen peroxide(Busciglio, J. & Yankner, B. A., Nature 378:776 (1995)). Synthetic ADpeptides have been shown to induce lipid peroxidation of synaptosomes(Butterfield, D. A., et al., Biochem. Biophys. Res. Commun. 200:710(1994)), and to exert neurotoxicity (Behl, C., et al., Cell 77:817(1994); Mattson, M. P., et al., J. Neurochem. 65:1740 (1995)) orvascular endothelial toxicity through a mechanism that involves thegeneration of cellular superoxide/hydrogen peroxide (O₂ ⁻/H₂O₂) and isabolished by the presence of SOD (Thomas, T., et al., Nature 380:168(1996) or catalytic synthetic O₂ ⁻/H₂O₂ scavengers (Bruce, A. J., etal., Proc. Natl. Acad. Sci. USA 93:2312 (1996)). Antioxidant vitamin Eand the spin-trap compound PBN have been shown to protect againstAβ-mediated neurotoxicity in vitro (Goodman, Y., & Mattson, M. P., Exp.Neurol. 128:1 (1994); Harris, M. E., et al., Exp. Neurol. 131:193(1995)).

[0297] Aβ, a 39-43 amino acid peptide, is produced (Haass, C., et al.,Nature 359:322 (1992); Seubert, P., et al., Nature 359:325 (1992);Shoji, M., et al., Science 258:126 (1992)) by constitutive cleavage ofthe amyloid protein precursor (APP) (Kang, J., et al., Nature 325:733(1987); Tanzi, R. E., et al., Nature Genet (1993)) as a mixture ofpolypeptides manifesting carboxyl-terminal heterogeneity. Aα-40 is themajor soluble Aβ species in biological fluids (Vigo-Pelfrey, C., et al.,J. Neurochem. 61:1965 (1993)) and Aβ₁₋₄₂ is a minor soluble species, butis heavily enriched in interstitial plaque amyloid (Masters, C. L., etal., Proc. Natl. Acad. Sci. USA 82:4245 (1985); Kang, J. et al., Nature325:733 (1987); Prelli, F., et al., J. Neurochem. 51:648 (1988); Roheret al., J. Cell Biol. 107:2703-2716 (1988); Roher et al., J. Neurochem.61:1916-1926 (1993); Miller, D. L., et al., Arch. Biochem. Biophys.301:41 (1993)). The discovery of pathogenic mutations of APP close to orwithin the Aβ domain (van Broeckhoven, C., et al., Science 248:1120(1990); Levy, E., et al., Science 248:1124 (1990); Goate, A., et al.,Nature 349:704 (1991); Murrell, J., et al., Science 254:94 (1991);Mullan, M., et al., Nature Genet 1:345 (1992)) indicates that themetabolism of Aβ is involved with the pathophysiology of thispredominantly sporadic disease. Familial AD-linked mutations of APP,presenilin-1 and presenilin-2 correlate with increased cortical amyloidburden and appear to induce an increase in the ratio of Aβ₁₋₄₂ as partof their common pathogenic mechanism (Suzuki, N., et al., Science264:1336 (1994); Scheuner et al., Nat Med., 2(8):864-870 (1996); Citron,M., et al., Nature Medicine 3:67 (1997)). However, the mechanism bywhich Aβ₁₋₄₂ exerts more neurotoxicity than Aβ₁₋₄₀ and other Aβ peptides(Dore, S., et al., Proc. Natl. Acad. Sci. USA 94:4772 (1997)) remainsunclear.

[0298] One of the models proposed for Aβ neurotoxicity is based on aseries of observations of Aβ-generated oxyradicals generated by aputative Aβ peptide fragmentation mechanism which is O₂-dependent,metal-independent and involves the sulfoxation of the methionine at Aβresidue 35 (Butterfield, D. A., et al., Biochem. Biophys. Res. Commun.200:710 (1994); Hensley, K., et al., Proc. Natl Acad. Sci. USA 91:3270(1994); Hensley, K., et al., Ann N Y Acad Sci., 786: 120-134 (1996).Aβ₂₅₋₃₅ peptide has been reported to exhibit H₂O₂-like reactivitytowards aqueous Fe²⁺, nitroxide spin probes, and synaptosomal membraneproteins (Butterfield, D. A., et al., Life Sci. 58:217 (1996)), andAβ₁₋₄₀ has also been reported to generate the hydroxyl radical bymechanisms that are unclear (Tomiyama, T., et al., J. Biol. Chem.271:6839 (1996)). However, there has been no quantitative appraisal ofthe ROS-generating capacity of Aβ₁₋₄₂ versus that of Aβ₁₋₄₀ and other Aβvariants, to date.

[0299] Aβ is a metal binding protein which saturably binds zinc via ahistidine-mediated specific high affinity site (K_(D)=107 nM) as well asby low affinity binding (K_(D)=5.2 μM). The high-affinity zinc bindingsite was mapped to a stretch of contiguous residues between positions6-28 of the Aβ sequence (Bush, A. I., et al., J. Biol. Chem. 269:12152(1994)). Concentrations of zinc ≧1 μM rapidly induce aggregation ofhuman Aβ₁₋₄₀ solutions (Bush, A. I., et al., Science 265:1464 (1994)),in reversible manner which is dependent upon the dimerization of peptidein solution, its alpha-helical content, and the concentration of NaCl(Huang, X., et al., J. Biol. Chem. 272:26464-26470 (1997)). Rat/mouseAβ₁₋₄₀ (“rat Aβ”, with substitutions o R₅→G, Y₁₀→F, and H₁₃→R, ascompared to human Aβ) binds zinc less avidly (a single binding site,K_(A)=3.8 μM) and, unlike the human peptide, is not precipitated by zincat concentrations ≦25 μM. Since zinc is concentrated in the neocortex,we hypothesized that the differential solubility of the rat/mouse Aβpeptide in the presence of zinc may explain the scarcity with whichthese animals form cerebral Aβ deposits (Johnstone, E. M., et al., Mol.Brain Res. 10:299 (1991); Shivers, B. D., et al., EMBO J. 7:1365(1988)).

[0300] We have also observed interactions of Aβ with Cu²⁺, whichstabilizes dimerization of Aβ₁₋₄₀ on gel chromatography (Bush, A. I., etal., J. Biol. Chem. 269:12152 (1994)), and which binds to the peptidewith an affinity estimated to be in the low picomolar range. Fe²⁺ hasbeen observed to induce partial aggregation of Aβ (Bush, A. I., et al.,Science 268:1921 (1995)), and to induce SDS-resistant polymerization ofthe peptide (Dyrks, T., et al., J. Biol. Chem. 267:18210-18217 (1992)).We hypothesized that the interactions of Aβ with Fe and Cu maycontribute to the genesis of the oxidation insults that are observed inthe AD-affected brain. This is because Fe³⁺ and Cu²⁺ are redox-activemetal ions that are concentrated in brain neurons, and may participatein the generation of ROS by transferring electrons in their reducedstate (reviewed in Markesbery, 1997).

[0301] The levels of Cu and Fe, and their binding proteins, aredysregulated in AD (Diebel, M. A., et al., J. Neurol. Sci. 143:137(1996); Good, P. F., et al., Ann. Neurol. 31:286 (1992); Robinson, S.R., et al., Alzheimer's Research ;1:191 (1995); Thompson, C. M., et al.,Neurotoxicology 9:1 (1988); Kennard, M. L., et al., Nature Medicine2:1230 (1996); Connor, J. R., et al., Neurosci. Lett. 159:88 (1993)) andmay therefore lead to conditions that could promote ROS production.While a direct role for Aβ in metal-dependent ROS generation has notbeen described, the peptide's physiochemical interation with transitionmetals, the presence of ferritin (Grudke-Iqbal, I., et al., ActaNeuropathol. 81:105 (1990)) and redox reactive iron (Smith, M. A., etal., Proc. Natl. Acad. Sci. USA 94:9866 (1997)) in amyloid lesions, andthe facilitation of Aβ₁₋₄₀ neurotoxicity in cell culture by nanomolarconcentrations of iron (Schubert, D. & Chevion, M., Biochem. Biophys.Res. Commun. 216:702 (1995)), collectively support such a possibility.

[0302] We report the simultaneous production of H₂O₂ and reduced metalions by Aβ, with the consequent generation of the hydroxyl radical. Theamounts of reduced metal and ROS were both greatest when generated byAβ₁₋₄₂>Aβ₁₋₄₀>>rat Aβ₁₋₄₀, Aβ₄₀₋₁ and Aβ₁₋₂₈, a chemical relationshipthat correlates with the relative neurotoxicity of these peptides. Thesedata describe a novel, O₂ ⁻ and biometal-dependent pathway of ROSgeneration by Alzheimer Aβ peptides which may explain the occurrence ofoxidative stress in AD brain.

[0303] a) Metal Ion Reduction by Aβ Peptides

[0304] To determine whether Aβ peptides possess metal-reducingproperties, the ability of Aβ peptides (Example 1) to reduce Fe³⁺ andCu²⁺, compared to Vitamin C and other polypeptides (Example 2) wasmeasured. Vitamin C, serving as a positive control, reducedCu²⁺efficiently (FIG. 13A). However, the reduction of Cu²⁺ by Aβ₁₋₄₂ wasas efficient, reducing all of the available Cu²⁺ during the incubationperiod. Aβ₁₋₄₀ reduced 60% of the available Cu²⁺, whereas rat Aβ₁₋₄₀ andAβ₁₋₂₈ reduced no Cu²⁺. The reduction of Cu²⁺ by BSA (25%) and insulin(10%) was less efficient than that by the human Aβ peptides, and was notunexpected since these polypeptides possess cysteine residues and reduceCu²⁺ in the process of forming disulfide bonds.

[0305] Fe³⁺/Fe²⁺ has lower standard reduction potential (0.11 V) thanCu²⁺/Cu⁺ (0.15 V) does under our experimental conditions (Miller, D. M.,et al., Free Radical Biology & Medicine 8:95 (1990)), and, in general,Fe³⁺ was reduced with less efficiency by Vitamin C and the polypeptidesthat reduced Cu²⁺. Vitamin C reduced 15% of the available Fe³⁺, howeverAβ₁₋₄₂ was the most efficient (50%) of the agents tested for Fe³⁺reduction, reducing more Fe³⁺ in the incubation period than Vitamin C(15%), Aβ₁₋₄₀ (12%) and BSA (8%). Rat Aβ₁₋₄₀, Aβ₁₋₂₈ and insulin did notsignificantly facilitate the reduction of Fe³⁺. Analysis of Aβ₁₋₄₂ andAβ₁₋₄₀ after incubation with Cu²⁺ and Fe³⁺ under these conditionsrevealed that there was no apparent mass modification of the peptides onmass spectrometry, and no change in its migration pattern on sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), norevidence for increased aggregation of the peptides by turbidometry orsedimentation analysis, suggesting that the peptides were not consumedor modified during the reduction reaction. Under these conditions, thecomplete kinetics of the peptide-mediated reactions cannot beappreciated (the presence of Aβ₁₋₄₂ induced the total consumption of theCu²⁺ substrate within the incubation period), but a strikingrelationship exists between the relative efficiencies of the various Aβpeptides to reduce Cu²⁺/Fe³⁺ in this system and their respectiveparticipation in amyloid neuropathology.

[0306] Since the dissolved O₂ in the buffer vehicle may be expected toreact with the reduced metals being generated [Reaction (1)], the effectof modulating the O₂ tension in the buffer upon the generation ofreduced metals by the Aβ peptides (FIG. 13R) was examined. Prior to theaddition of Vitamin C or polypeptide, the buffer vehicle wascontinuously bubbled for 2 hours at 20° C. with 100% O₂ to createconditions of increased O2 tension, or Argon to create anaerobicconditions. Increasing the O₂ tension slightly reduced the levels ofreduced metals being detected, probably due to the diversion of afraction of the Fe²⁺/Cu⁺ being generated to Reaction (1), and, if H₂O₂is being produced as a product of Reaction (2), the recruitment ofFe²⁺/Cu⁺ into the Fenton reaction [Reaction (3)]. However, performingthe reaction under anaerobic (Argon purged) conditions also slightlyreduced the levels of reduced metals being detected. This may be becausesome of the reduction of Fe³⁺/Cu²⁺ is due to reaction with O₂ ⁻:

M^((n+1)+)O₂ ⁻→M^(n+)+O₂  Reaction (5)

[0307] To determine whether the reduction of metal ions in the presenceof Aβ was due to the action of the peptide or the generation of O²⁻ bythe peptide, the effects of metal ion chelators on the generation ofreduced metal ions (FIG. 13B) was studied. It was found thatcoincubation of Aβ₁₋₄₂ with the relatively Fe³⁺-specific chelatordesferrioxamine (DFO) under ambient oxygenation conditions nearly halvedthe production of Fe²⁺. Coincubation of Aβ₁₋₄₂ with the high-affinityCu²⁺ chelator TETA abolished 95% of the Cu⁺ generated by the peptideunder ambient oxygenation conditions. These data indicate that themajority of the Cu⁺ and a significant amount of the Fe²⁺ produced byAβ₁₋₄₂ are due to the direct action of the peptide and not indirectlydue to the production of O₂ ⁻.

[0308] The inhibitory effects of chelation upon Aβ-mediated reduction ofmetal ions indicates that Aβ probably directly coordinates Fe³⁺ andCu²⁺, and also that these chelating agents are not potentiating theredox potential of the metals ions, suggested to be an artifactualmechanism for the generation of reduced metal species (Sayre, L. M. etal., Science 274:1933 (1996)). The reasons for DFO being less effectivethan TETA in attenuating metal reduction may relate to the respective(unknown) binding affinities for Fe³⁺ and Cu²⁺ to the Aβ peptide, thestereochemistry of the coordination of the metal ions by the peptide,and the abilities of the chelating agents to affect electron transferafter coordinating the metal ion.

[0309] The reduction of metal ions by Aβ must leave the peptide, atleast transiently, radicalized, in agreement with the electronparamagnetic resonance (EPR) findings of Hensley et al., Proc. Natl.Acad. Sci. 91:3270 (1994). In their report, DFO, EDTA or Chelex 100could not abolish the EPR signal generated by Aβ₂₅₋₃₅ in PBS, leadingthese investigators to conclude that the radicalization of Aβ wasmetal-independent. However, the inventors have found that aftertreatment with Chelex 100 the concentrations of Fe and Cu in PBS arestill as high as ≈0.5 μM (8), which could be sufficient to induce theradicalization of the peptide after metal reduction. Since DFO does notabolish the reduction of Fe³⁺ by Aβ₁₋₄₂ (FIG. 13B), and since EDTA hasbeen observed to potentiate Fe-mediated Fenton chemistry (Samuni et al.,Eur. J. Biochem. 137:119-124(1983)), it is suspected that Hensley andcolleagues may have inadvertently overlooked the contribution of metalreduction to Aβ-mediated radical formation.

[0310] Rat Aβ₁₋₄₀ did not reduce metal ions, and has been shown to haveattenuated binding of Zn²⁺ (Bush et al., Science, 265:1464 (1994)). Asimilar attenuation of Cu²⁺ and Fe³⁺ binding by rat Aβ₁₋₄₀ compared tohuman Aβ₁₋₄₁ is anticipated. These data also indicate that the rat Aβsubstitutions in human Aβ's zinc binding domain towards the peptide'samnino terminus (Bush et al., J. Biol. Chem., 269:12152 (1994)) involveresidues that mediate the metal-reducing properties of the peptide.However, the hydrophobic carboxyl-terminal residues were also criticalto the reduction properties of Aβ. That Aβ₁₋₂₈ did not reduce metal ionsindicates that an intact Zn²⁺-binding site (Bush et al., J. BioL. Chem.269:12152 (1994)) is insufficient to facilitate the metal reductionreaction. The mechanism by which the two additional hydrophobic residues(Ile and Ala) on Aβ₁₋₄₂ so substantially enhance the peptide's redoxactivity compared to Aβ₁₋₄₀ is still unclear.

[0311] It has been observed that sulfoxation of the methionine residueat AD position 35 accompanies the EPR changes seen during the incubationof Aβ₂₅₋₃₅ for 3 hours in PBS at 37° C. (Hensley, K., et al., Ann N YAcad Sci., 786: 120-134 (1996)), however, no evidence was found for amodification of Aβ₁₋₄₀ and Aβ₁₋₄₂ afier mass spectrophotometricexamination of the peptides incubated under conditions as described.Therefore, Aβ-mediated metal reduction, and the subsequent Aβ-mediatedredox reactions described below, appear to be achieved by a mechanismthat differs from that previously reported.

[0312] b) Production of H₂O₂ by Aβ Peptides

[0313] The reduced metal ions produced by Aβ were expected to generateO₂ and H₂O₂ by Reactions (1) and (2). To study this, a novel assay wasdeveloped (Example 3) which detected the generation of 10 μM H₂O₂ byAβ₁₋₄₂ in the presence of 1 μM Fe³⁺ under ambient O₂ conditions (FIG.14A). To validate the assay, coineubation with eatalase was observed toabolish the H₂O₂ signal in a dose dependent manner. The amount of H₂O₂produced by the various Aβ peptides was studied, and observed that theorder of the production of H₂O₂ by the Aβ variants wasAβ₁₋₄₂>Aβ₁₋₄₀,>rat Aβ₁₋₄₀- Aβ₁₋₂₈ (FIG. 14B), paralleling the amounts ofmetal reduction by the same peptides (FIG. 13A).

[0314] H₂O₂ formation is likely to be mediated first by O₂-dependent O₂⁻ formation [Reaction (1)], followed by dismutation [Reaction (2)]. Toappraise the contribution of Reaction (1) to H₂O₂ formation, H₂O₂formation by Aβ₁₋₄₂ in the presence of chelators was measured (FIG.14C). The amount of H₂O₂ formed in the presence of 1 μM Cu²⁺ was 25%greater than the amount formed in the presence of 1 μM Fe³⁺.Coincubation with DFO had no effect on H₂O₂ formation in the presence of1 μM Fe³⁺. However, TETA, and the Cu⁺-specific indicator BC, bothsubstantially inhibited the formation of H₂O₂ in the presence of 1 μMCu²⁺. The reasons why DFO partially inhibited Fe³⁺ reduction, but wasunable to inhibit H₂O₂ formation are unclear. These data indicate thatthe formation of H₂O₂ by Aβ is dependent upon the presence ofsubstoichiometric amounts of Cu⁺/(II). The possibility that formation ofH₂O₂ in the presence of Fe³⁺ was due to the presence of trace quantitiesof Cu²⁺ cannot be excluded.

[0315] BC and BP, agents that specifically complex reduced metal ions,were far more effective than DFO and TETA at inhibiting H₂O₂ formationby Aβ (FIG. 14C) but the reasons for this are not clear. The relativelyFe²⁺-specific complexing agent, BP, inhibited H₂O₂ formation in thepresence of Cu²⁺, and the relatively Cu⁺-specific complexing agent, BC,inhibited H₂O₂ formation in the present of Fe³⁺, suggesting that theseagents are not totally specific in their metal ion affinities. Theformation of H₂O₂ by Aβ in the absence of BC or BP confirms that thereduction of metals is not contingent upon the artifactual enhancementof the metal ions' redox potentials (Sayre, L. M., Science 274:1933(1996)).

[0316] To determine whether the formation of O₂ ⁻/H₂O₂ by Aβ is merelydue to the reduction of metal ions, or whether Aβ also facilitates therecruitment of the substrates in Reaction (1), the generation of H₂O₂ byAβ₁₋₄₂, Aβ₁₋₄₀ and Vitamin C under different O₂ tensions in the presenceof 1 μM Fe³⁺ (FIG. 14D) or 1 μM Cu²⁺ (FIG. 14E) was studied. Thepresence of Vitamin C was used as a control measure to determine theamount H₂O₂ that is generated by the presence of reduced metals alone.In the presence of either metal ion, there was a significant increase inthe amount of H₂O₂ produced under higher O₂ tensions. The presence ofeither Aβ₁₋₄₂ and Aβ₁₋₄₀ generated more H₂O₂ (Aβ₁₋₄₂ >A⊕₁₋₄₀) thanVitamin C under any O₂ tension studied, and generated H₂O₂ underconditions where Vitamin C produced none, even though reduced metal ionsmust be present due to the activity of Vitamin C. Therefore, under theseambient and argon-purged conditions, the reduction of metal ions isinsufficient to produce H₂O₂. These data indicate that Aβ indeedfacilitates the recruitment of O₂ into Reaction (1) more than would beexpected by the interaction of the metals reduced by Aβ with thepassively dissolved O₂. Under relatively anaerobic conditions, the Aβpeptides were observed to still produce H₂O₂ in the presence of Cu²⁺(FIG. 14E). This is probably due to the ability of Aβ to recruit O₂ intoReaction (1) under conditions of very low O₂ tension. Since O₂ ispreferentially dissolved in hydrophobic environments (Halliwell andGutteridge, Biochem. J., 219:1-14 (1984)), it seems that the hydrophobiccarboxyl-terminus of Aβ could attract O₂, serving as a reservoir for thesubstrate.

[0317] c) Evidence of the Superoxide Anion Formed by tile Aβ-metalComplex

[0318] To confirm the production of O₂ ⁻ by Aβ, the absorbance of thepeptide in solution at 250 nm, the absorbance peak of O₂ ⁻ (FIG. 15A)was measured. The absorbance generated by Aβ₁₋₄₂ in the presence of 1 μMFe³⁺ was 60% reduced when co-incubated with SOD, increased in thepresence of high O₂ tension and abolished under anaerobic conditions.These data support the likelihood that Aβ generates H₂O₂ by firstgenerating O₂ ⁻.

[0319] The absorbance changes at 250 nm for the various AD peptides inPBS (FIG. 15B) paralleled the production of H₂O₂ from the same peptides(FIG. 14B), but the reason for the A₂₅₀ being much greater for Aβ₁₋₄₂compared to Aβ₁₋₄₀ is unclear. It is likely that a fraction of the totalH₂O₂ generated by Aβ is decomposed by the Fenton reaction [Reaction(3)]. Therefore, the amount of H₂O₂ detected may be an attenuatedreflection of the amount of O₂ ⁻ detected.

[0320] d) Detection of Hydroxyl Radicals Generatedfrom the Aβ-metalComplex

[0321] Having demonstrated that human Aβ peptides simultaneously produceH₂O₂ and reduced metals, it was determined whether the hydroxyl radicalwas formed by the Fenton or Haber-Weiss reactions [Reactions (3) and(4)]. A modified TBARS assay was employed to detect OH released fromco-incubation mixtures of Aβ peptides and 1 μM Fe³⁺ or Cu²+. Asexpected, Aβ₁₋₄₂ produced more OH than Aβ₁₋₄₀, and rat Aβ did notgenerate OH (FIG. 16A). In contrast to the amount of Fe²⁺ and Cu⁺produced (FIG. 13A), Aβ generated more OH. in the presence of Fe³⁺ thanin the presence of Cu²⁺. This may be because Fe²⁺ is more stable thanCu⁺, which may be more rapidly oxidized by Reaction (1). Therefore, theFe²⁺ generated by Aβ may have a greater opportunity than the Cu⁺generated to react with H₂O₂. It is also possible that the contributionof the Haber-Weiss reaction to the production of OH. [Reaction (5)] isgreater in the presence of Fe³⁺ than in the presence of Cu²⁺.

[0322] The effects of the OHS scavengers, dimethyl sulfoxide (DMSO) andmannitol, upon Aβ₁₋₄₂-mediated OHS generation were studied. Whereasthese agents suppressed the generation of OH. by Vitamin C in thepresence of Fe and DMSO suppressed the generation of OH by Vitamin C inthe presence of Cu²⁺, neither were able to quench the generation of OH.by Aβ₁₋₄₂, whether in the presence of Fe³⁺ or Cu²⁺ (FIG. 16B). Thissuggests that these scavengers cannot encounter the OH. generated by Aβbefore the TBARS reagent does.

[0323] e) Similarity Between Bleomycin-Fe and Aβ-Fe/Cu Complexes

[0324] The present Examples provide evidence for a model by which Fe/Cuand O₂ are mediators and substrates for the production of OH. by Aβ(FIGS. 16A and 16B) in a manner that depends upon the presence andlength of the peptide's carboxyl terminus. The brain neocortex is anenvironment that is rich in both O₂ and Fe/Cu, which may explain whythis organ is predisposed to Aβ-mediated neurotoxicity, if thismechanism is confirmed in vivo. The transport of Fe, Cu and Zn in thebrain is largely energy-dependent. For example, the copper-transportinggene for Wilson's disease is an ATPase (Tanzi, R. E. et al., NatureGenetics 5:344 (1993)), and the re-uptake of zinc followingneurotransmission is highly energy-dependent (Assaf, S. Y. & S. H.Chung, Nature, 308:734-736 (1984); Howell et al., Nature, 308:736-738(1984)).

[0325] There is increasing evidence for lesions of brain energymetabolism in aging and AD (Parker et al., Neurology, 40:1302-1303(1990); (Mecocci et al., Ann. Neurol. 34:609-616 (1993); Beal, M. F.Neurobiol. Aging 15 (Suppl 2):S171-S174(1994)). Therefore, damage toenergy-dependent brain metal homeostasis may be an upstream lesion forthe genesis of Aβ deposition in AD. Most brain biometals are bound toproteins or other ligands, however, according to our findings, only Aβsmall fraction of the available metals needs to be derailed to theAβ-containing compartment to precipitate the peptide and to activate itsROS-generating activities. The generation of ROS described hereindepends upon the sub-stoichiometric amounts of Fe⁺/Cu2+ (1:10,metal:Aβ), and it was estimated that 1% of the zinc that is releasedduring neurotransmission would be sufficient to precipitate soluble Aβin the synaptic vicinity (Huang, X. et al., J. Biol. Chem.272:26464-26470 (1997)).

[0326] A polypeptide which generates both substrates of the Fentonreaction in sufficient quantities to form significant amounts of the OH.radical is unusual. Therefore, Aβ collections in the AD-affected brainare likely to be a major source of the oxidation stress seen in theeffected tissue. One recent report describes that Aβ is released by thetreatment of the mammalian lens in culture with H₂O₂ (Frederikse, P. H.,et al., J. Biol. Chem. 271:10169 (1996)). If a similar responsemechanism to H₂O₂ stress exists in neocortex, then the increasing H₂O₂concentration generated by the accumulating Aβ mass in the AD-affectedbrain may induce the production of even more Aβ leading to a viciouscycle of Aβ accumulation and ROS stress.

[0327] The simultaneous production of Fenton substrates by Aβ is achemical property that is brought into therapeutic application in theoxidation mechanism of the bleomycin-iron complex. Bleomycin is aglycopeptide antibiotic produced by Streptomyces verticillus and is apotent antitumor agent. It acts by complexing Fe³⁺ and then binding totumor nuclear DNA which is degraded in situ by the generation of OHS(Sugiura, Y., et al., Biochem. Biophys. Res. Commun. 105:1511(1997)).Similar to Aβ-Fe³⁺/Cu²⁺ complexes, incubation of bleomycin in aqueoussolution also engenders the production of O₂ ⁻, H₂O₂ and OH. in anFe⁺-dependent manner. DFO could not inhibit H₂O₂ production from theAβ-Fe⁺/Cu²⁺ complex, and similarly, DFO does not inhibit theOH.-mediated DNA damage caused by the bleomycin-Fe³⁺ complex. Also,low-molecular-mass OH. scavengers mannitol and DMSO were unable toinhibit the generation of OH. by Aβ-Fe³⁺/Cu²⁺, and are similarly unableto inhibit OH. production from bleomycin-Fe³⁺.

[0328] It is proposed herein that inhibition of Aβ-mediated OHS providesmeans of treatment, e.g. therapy, by compounds that are Fe or Cuchelators. The clinical administration of DFO was reported as beingeffective in preventing the progression of AD (Crapper-McLachlan, D. R.et al., Lancet 337:1304 (1991)); however, since DFO chelates Zn²⁺ aswell as Fe³⁺ and Al(III), the effect, if verifiable, may not have beendue to the abolition of the redox activity of Aβ, but may have been dueto the disaggregation of Zn²⁺-mediated Aβ deposits (Chemy, R. A. et al.,Soc. Neurosci. Abstr. 23:(abstract)(1997)) which may have reducedcortical Aβ burden and, consequently, oxidation stress.

[0329] f) Oxidative Stress and Alzheimer's Disease Pathology

[0330] Autopsy tissue from AD subjects has been reported to exhibithigher basal TBARS formation than control material (Subbarao, K. V. etal., J. Neurochem. 55:342 (1990); Balazs, L. and M. Leon, Neurochem.Res. 19:1131 (1994); Lovell et al., Neurology 45:1594 (1995)). Theseobservations could be explained, on the basis of the present findings,as being due to the reactivity of the Aβ content within the tissue.Aβ₁₋₄₀ recently has been shown to generate TBARS in a dose-dependentmanner when incubated in cell culture, however TBARS reactivity wasreduced by pre-treating the cells with trypsin which also abolished thebinding of the peptide to the RAGE receptor (Yan et al., Nature 382:685(1996)). One possibility for this result is that the RAGE receptortethers an Aβ microaggregate sufficiently close to the cell to permitincreased penetration of the cell by H₂O₂ which may then combine withreduced metals within the cell to generate the Fenton reaction.Alternatively, Aβ may generate the Fenton chemistry at the RAGEreceptor. The resulting attack of the cell surface by the highlyreactive OH. radical, which reacts within nanometers of its generation,may have been the source of the positive TBARS assay.

[0331] APP also reduces Cu²⁺, but not Fe³⁺, at a site in its aminoterminus (Multhaup, G., et al., Science 271:1406-1409 (1996)), adjacentto a functional and specific Zn²⁺-binding site that modulates heparinbinding and protease inhibition (Bush et al., 1993; Van Nostrand, 1995).Therefore, the amino terminus of APP reiterates an association withtransition metal ions that is found in the Aβ domain. This intriguingtheme of tandem Cu/Zn interaction and associated redox activity found intwo soluble fragments of the parent protein may indicate that thefunction and metabolism of APP could be related to biometal homeostasisand associated redox environments.

[0332] The present findings indicate that the manipulation of the brainbiometal environment with specific agents acting directly (e.g.chelators and antioxidants) or indirectly (e.g. by improving cerebralenergy metabolism) holds promise as a means for therapeutic interventionin the prevention and treatment of Alzheimer's disease.

Example 4 Resolubilization of Aβ

[0333] Considerable evidence now indicates that the accumulation of Aβin the brain cortex is very closely related to the cause of Alzheimer'sdisease. Aβ is a normal component of biological fluids whose function isunknown. Aβ accumulates in a number of morphologies varying from highlyinsoluble amyloid to deposits that can be extracted from post-mortemtissue in aqueous buffer. The factors behind the accumulation areunknown, but the inventors have systematically appraised the solubilityof synthetic Aβ peptide in order to get some clues as to what kind ofpathological environment could induce the peptide to precipitate.

[0334] It was found that Aβ has three principal vulnerabilities—zinc,copper and low pH. The precipitation of Aβ by copper is dramaticallyexaggerated under mildly acidic conditions (e.g., pH 6.9), suggestingthat the cerebral lactic acidosis that complicates Alzheimer's diseasecould contribute to the precipitation of AD were this event to bemediated by copper. A consideration of the involvement of zinc andcopper in plaque pathology is contemplatable since the regulation ofthese metals in the brain has been shown to be abnormal in AD.

[0335] Recently direct evidence has been obtained indicating that thesemetals are integral components of the Aβ deposits in the brain in AD. Itwas found that zinc- and copper-specific chelators (includingclioquinol) dramatically redissolve a significant proportion (up to 70%)of Aβ extracted from post-mortem AD affected brain tissue, compared tothe amount extracted from the tissue by buffer in the absence ofchelators.

[0336] These data support a strategy of redissolving Aβ deposits in vivoby chelation. Therefore, clioquinol is an excellent candidate forfurther development since it chelates both copper and zinc, and since itis hydrophobic, is enriched in the brain. Interestingly, a reportedsuccess in attempting to slow down the progression of Alzheimer'sdisease used a chelation strategy with desferrioxamine. The authors(Crapper-McLachlan, D. R., et al., 337:1304 (1991), thought that theywere chelating aluminum, but desferrioxamine is also a chelator ofcopper and zinc. Treatment with desferrioxamine is impractical becausethe therapy requires twice daily deep intramuscular injections which arevery painful, and also causes side effects such as anaemia due to ironchelation.

[0337] Resolubilization of Metal-induced Aβ Aggregates by Chelators

[0338] Aβ (10 ng/well in TBS) aggregation was induced by addition ofZnCl₂ (25 μM), CuCl₂ (5 μM) or acidic conditions (pH 5.5). Aggregateswere transferred to a 0.2 μ nylon membrane by filtration. The aggregateswere then washed (200 μl/well) with TBS alone, TBS containing 2 μM EDTA,or TBS containing 2 μM clioquinol. The membrane was fixed, probed withthe anti-Aβ monoclonal antibody 6E10, and developed for exposure toECL-film. FIG. 17 shows relative signal strength as determined bytransmittance analysis of the ECL-film, calibrated against known amountsof the peptide. Values are expressed as a percentage of Aβ signal afterwashing with TBS alone.

[0339] Both EDTA and clioquinol treatments were more effective than TBSalone at resolubilizing the retained (aggregated) Aβ when the peptidewas precipitated by Zn or Cu (see FIG. 17). When Aβ was precipitated bypH 5.5 however, it was not resolubilized more readily by either chelatorcompared to TBS washing alone. The pH 5.5 precipitate contains a muchgreater proportion of beta-sheet amyloid than the Aβ precipitates formedby Zn or Cu.

Example 5 Aβ Extractionfrom Human Brain Post-mortem Samples

[0340] The inventors have recently characterized zinc-mediated Aβdeposits in human brain (Cherny, R. A., et al., Soc. Neurosci 4bstr.23:(Abstract) (1997)). It was recently reported that there is apopulation of water-extractable Aβ deposit in the AD-affected brain(Kuo, Y -M., et al., J. Biol. Chem. 271:4077-81 (1996)). The inventorshypothesized that homogenization of brain tissue in water may dilute themetal content in the tissue, so lowering the putative zinc concentrationin Aβ collections, and liberating soluble Aβ subunits by freeing Aβcomplexed with zinc [Zn²⁺].

[0341] To test this hypothesis, the brain tissue preparation protocol ofKuo and colleagues was replicated, but phosphate-buffered saline pH 7.4(PBS) was substituted as the extraction buffer, achieving similarresults. Highly sensitive and specific anti-Aβ monoclonal antibodies(Ida, N., et al., J. Biol. Chem., 271:22908 (1996)) were used to assayAβ extraction by western blot. Next, the extraction of the same materialwas repeated with PBS in the presence of chelators of varyingspecificities (Table 1), and it was determined that the presence of achelator increased the amount of Aβ in the soluble extract several-fold(FIGS. 19A-19C, 20A and 20B, 25A; Table 2).

[0342] The amount of Aβ detected in the pellet fraction of each sampleis correspondingly lower, indicating that the effect of the chelator isupon the disassembly of the Aβ aggregate, and not by inhibition of anAβ-cleaving metalloprotease (such as insulin degrading enzyme cleavageof Aβ reported recently by Dennis Selkoe at the 27^(th) Annual Meetingfor the Society for Neuroscience, New Orleans). The extraction ofsedimentable Aβ into the soluble phase correlated only with theextraction of zinc from the pellet, and not with any other metal assayed(Table 3). Examination of the total amount of protein released by thetreatments revealed that chelation was not merely liberating moreproteins in a non-specific manner. TABLE 1 Dissociation Constants forMetal Ions of Various Chelators Used to Extract Human Brain Aβ. CHELATORCa Cu Mg Fe Zn Al Co EGTA 10.9 17.6 5.3 11.8 12.6 13.9 12.4 EDTA 10.718.8 8.9 14.3 16.5 16.5 16.5 Penicillamine 0 18.2 0 0 10.2 0 0 TPEN 3.020.2 0 14.4 15.4 0 0 Bathophenanthroline 0 8.8 0 5.6 6.9 0 0Bathocuproine (BC) 0 19.1 0 0 4.1 0 4.2 (Cu⁺)

[0343] LogK is illustrated for the chelators, where K=[ML]/[M][L].Different chelators have greatly differing affinities for metal ions, asshown. TPEN is relatively specific for Zn and Cu, and has no affinityfor Ca and Mg (which are far more abundant metal ions in tissues).Bathocuproine (BC) has high affinity for zinc and for cuprous ions.Whereas all the chelators examined have a significant affinity for zinc,EGTA and EDTA have significant affinities for Ca and Mg.

[0344] The ability of chelators to extract Aβ from post-mortem braintissue was studied in over 40 cases (25 AD, 15 age-matched and youngadult controls, all confirmed by histopathology). While there is a lotof variation between samples as to what is the best concentration ofgiven chelator for the optimum extraction of Aβ, there are no caseswhere a chelator does not, at some concentration, extract far more Aβthan PBS alone.

[0345]FIG. 19 shows that metal chelators promote the solubilization ofAβ from human brain sample homogenates. Representative curves for threechelators (TPEN, EGTA, Bathocuproine) used in extracting the samerepresentative AD brain sample are shown. 0.5 g of prefrontal cortex wasdissected and homogenized in PBS ±chelator as indicated. The homogenatewas then centrifuged (100,000 g) and the supernatant removed, and asample taken for western blot assay using anti-Aβ specific antibodiesafter Tricine PAGE. Densitometry was performed against synthetic peptidestandards. The blots shown here represent typical results. Similarresults were achieved whether or not protease inhibitors were includedin the PBS (extraction was at 4° C.). Furthermore, similar results wereachieved when the brain sample was homogenized in PBS and then pelletedbefore treated with PBS ±chelator.

[0346] There is also a complex relationship between the dose of thechelator and the resultant resolubilization of Aβ (FIGS. 19A-C). For thesame given sample, neither TPEN nor EGTA could increase the extractionof Aβ in a does-dependent manner. Rather, although concentrations ofchelators could be very effective in the low micromolar range (e.g.,TPEN 4 μM, FIG. 19A), higher concentrations induced a paradoxical lossof recovery. This kind of response was found in every case examined. Theextraction of Aβ is abolished by adding exogenous zinc, but is enhancedby adding magnesium. Preliminary in vitro data indicate that whereas Mghas no effect on the precipitation of Aβ, its presence enhances thepeptide's resolubilization following zinc-induced precipitation.Therefore, the “polyphasic” profile of chelator extraction of Aβ, withhigher concentrations of TPEN and EGTA inducing a loss of recovery, maybe explained by the chelation of Mg that is only expected to occur afterthe chelation of zinc when the relative abundance of Mg in the sample,and the relative dissociation constants of TPEN and EGTA are considered.

[0347] In contrast, bathocuproine (BC) exhibits a clear dose-dependentincrease in Aβ extraction from human brain, probably due to itsrelatively high specificity for zinc, although an interaction with traceamounts of Cu⁺ or other metals not yet assayed, cannot be excluded.

[0348] Western blot analysis of extracts using Aβ₁₋₄₂-specificmonoclonals revealed the presence of abundant Aβ₁₋₄₂ species. It wasobserved that ≈20% of AD cases exhibit clear SDS-resistant Aβ dimers inthe soluble extract after treatment with chelators. These dimers arereminiscent of the neurotoxic Aβ₁₋₄₂ dimers that were extracted by Roherand colleagues from AD-affected brain (Roher, A. E., et al., Journal ofBiological Chemistry 271:20631-20635 (1996)). An estimation of theproportion of total precipitated Aβ in the sample was achieved byextracting the homogenate pellet following centrifligation, into formicacid, and then performing a western blot on the extract followingneutralization. The proportion of pelletable Aβ that is released bychelation treatment varies considerably from case to case, from aslittle as 30% to as much as 80%. In the absence of a chelator, no morethan 10% of the total pelletable Aβ is extracted by PBS alone.

[0349] One preliminary emerging trend is that samples with a greaterproportion of diffuse or vascular Aβ deposit are more likely to havetheir pelletable Aβ resolubilized by chelation treatment. Also,extraction of the tissue homogenate overnight with agitation greatlyincreases the amount of Aβ extracted in the presence of chelators(compared to PBS alone), when compared to briefer periods of extractionindicating that the disassembly of Aβ deposits by chelation treatment isa time-dependent reaction and is unlikely to be due to inhibition of aprotease. A study of brain cortical tissue from one amyloid-bearing APPtransgenic mouse indicates that, like human brain, homogenization in thepresence of a chelator enhances the extraction of pelletable Aβ.

[0350] Effects of various chelators on the extraction of Aβ into thesupernatant as a percentage change from control extractions issummarized below in Table 2. TABLE 2 Effects of Various Chelators UponExtraction of Aβ. Effect of Chelators (% change from control) TPEN EGTABATHOCUP 0.1 mM 2.0 mM 0.1 mM 2.0 mM 0.1 mM 2.0 mM Mean (n = 6) 182 241207 46 301 400 +/−SD 79 81 115 48 190 181

[0351] Densitometry of Aβ western blots (FIGS. 19A-19C) was performedfor a series of 6 AD brain samples homogenized in the presence ofchelators as indicated. The mean (±SD) increases in signal, above thesignal generated by PBS extraction alone, are indicated in Table 2. Asignificantly increased amount of chelator-induced Aβ resolubilizationwas achieved by a 16 hour extraction with agitation in subsequentstudies.

[0352] Table 3 shows a comparison between pellets of post-centrifugationhomogenates in the presence and absence of a chelator (TPEN). TABLE 3Residual Metals in Pellets of Post-Centrifugation Homogenatesin thePresence and Absence of Chelator. METAL Zn Cu Fe Ca Mg Al PBS 50.7 11.9227 202 197 44 alone (12.0) (3.5) (69) (69) (94) (111) mg/kg (SD) +TPEN33.2* 9.8 239 (210) 230 65 mg/kg (9.8) (3.1) (76) (89) (94) (108) (SD)

[0353] Frontal cortex from AD (n=6) and healthy controls (n=4) washomogenized in the presence and absence of PBS i TPEN (0.1 mM). Afterultracentrifugation of the homogenate, the pellets were extracted intoconcentrated HCl and measured for metal content by ion coupledplasma—atomic emission spectroscopy (ICP-AES).

[0354] Using the same technique, zinc-mediated assembly of Aβ in normalbrains was shown. FIGS. 20A and 20B show sedimentable Aβ deposits inhealthy brain tissue. The effects of chelators in enhancing Aβextraction from brain homogenates is also observed in normal tissue.FIG. 20A illustrates a western blot with anti-Aβ antibody of materialextracted from a 27-year-old individual with no history of neurologicaldisorder. T=TPEN, E=EGTA, B=bathocuproine. Bathocuproine is much lesseffective in extracting Aβ from control tissue than from AD tissue.These data are typical of 15 cases.

[0355] As expected, far less total Aβ is present in normal brain samplescompared to AD brain samples, although the content of Aβ increases withage. It is possible that these findings in young adult brains representthe zinc-mediated initiation of amyloid formation in deposits that, inyouth, are too diffuse to be detected by immunohistochemistry.

[0356] Roher and others have suggested that dimers of Aβ are the toxiccomponent of amyloid. As shown in FIG. 21, dimers appear in response tochelation in disproportion to the monomeric signal (treatment with PBSalone does not generate soluble dimers). This suggests that Aβ depositsare being dismantled by the chelators into SDS-resistant dimericstructural units.

[0357]FIG. 22 shows that the recovery of total soluble protein is notaffected by the presence of chelators in the homogenization step. Theproportionality of extracted subfractions, calculated based on totalprotein as determined by formic acid extraction, should not be prone toartifact based on chelator-specific affects.

Example 6 Resolubilization of Aβ by Clioquinol

[0358] In one previous attempt to use metal chelation as a therapeuticfor AD, Crapper-McLachlan and colleagues (Crapper-McLachlan, D. R., etal., 337:1304 (1991)) administered intramuscular desferrioxamine (DFO)daily to a small cohort of AD patients, and reported that theirtreatment attenuated the progression of the disease. Replication of thisstudy has not been attempted.

[0359] The inventors attributed the beneficial effect to the removal ofaluminum; however, they have conceded in presentations at meetings (e.g.International Conference on Alzheimer's Disease, 1992, Padua) thatpost-mortem metal analysis on brain tissue from subjects in the studyindicated that although aluminum levels were lowered than placebocontrols, zinc and iron levels were also lower in the brains of subjectstreated with DFO. This is because, like all chelators, DFO has only arelative specificity for aluminum, but with also complex with zinc andiron. There appears to be no report on a histopathological analysis ofpost-mortem brain Aβ content in the subjects who took DFO compared tothe controls.

[0360] The administration of DFO, a painful intramuscular injection, isfraught with complications including the non-specific problems ofchelation therapies (e.g. anemia). Although the results ofCrapper-McLachlan and colleagues remain contentious and have not yetbeen reproduced, the possibility that the beneficial effects theyreported were due to the partial removal of zinc from brain Aβcollections cannot be excluded. DFO is a charged molecule that does noteasily penetrate the blood-brain barrier, and, as such, is not an idealcandidate for the removal of zinc from Aβ deposits, especially as itsaffinity for zinc is relatively low. Therefore, a more suitablecandidate compound to attempt a trial of Aβ Dissolution in APP Tgs wassought.

[0361] Clioquinol (iodochlorhydroxyquin,5-chloro-7-iodo-8-hydroxyquinoline, MW 305.5) is a USP drug thatchelates zinc [K(Zn)=12.5, K(Cu)=15.8, K(Ca)=8.1, K(Mg)=8.6], ishydrophobic, has a low general toxicity profile, and crosses the bloodbrain barrier (Padmanabhan et al., 1989). It therefore possesses some ofthe ideal prototypic properties for a candidate agent that couldsolubilize zinc-assembled Aβ deposits in vivo. It has been used as anoral antiamebic antibiotic, and as a topical antibiotic.

[0362] It has been demonstrated that clioquinol is rapidly absorbed fromthe gut of rats and mice where blood levels reached 1-10 μM within onehour of ingestion (Kotaki et al., J Pharmacobiodyn, 6(11):881-887(1983)). Since the drug is hydrophobic, it passes rapidly into thebrain, and then is rapidly excreted, so that a bolus dose of clioquinolis almost completely removed from the brain within three hours. Itappears to be safe in many mammalian species, including rat and mouse(Tateishi, J., et al., Lancet, 2(7786):1096 (1972); Tateishi, J., etal., Acta Neuropathol., (Berl), 24(4):304-320 (1973)), and is still usedas a veterinary antibiotic (Entero Vioform).

[0363] Clioquinol was withdrawn from use as an oral antibiotic forhumans in the early 1970's when its ingestion in Japan was linked to amysterious condition called subacute myelo-optic neuritis (SMON), acondition that resembles subacute combined degeneration of the cordcaused by vitamin B12 deficiency. The mechanism of SMON has never beenelucidated, but in the 1970's a considerable literature developedexploring the pathophysiology of clioquinol ingestion (Tateishi, J., etal., Lancet, 2(7786):1096 (1972); Tateishi, J., et al., ActaNeuropathol., (Berl), 24(4):304-320 (1973)). Several reports havedemonstrated that clioquinol complexes with zinc in the brain,especially in areas enriched in synaptic vesicular zinc such as thetemporal lobe (Shiraki, H. Handbook of Clinical Neurology, Vol. 37(1979)). Indeed, over ingestion of clioquinol has been reported toinduce amnesia in humans (Shiraki, H. Handbook of Clinical Neurology,Vol. 37 (1979)).

[0364] Because of its relatively safe profile in mice, and because thereis a large literature on its pharmacology in this animal, clioquinol waschosen for study as a means to specifically chelate zinc from Aβdeposits in vitro (induced aggregates and brain samples). It is possiblethat the low concentrations of clioquinol shown to be effective inresolubilizing Aβ in the present invention may avoid the adverse SMONeffect noted above. Thus, given its other pharmacological properties,clioquinol may hold promise as a effective agent in the treatment of ADin humans.

[0365] Dissolving Clioquinol

[0366] In order to obtain a solution of clioquinol in PBS, the followingprotocol was followed: 5.3 grams of clioquinol was suspended withagitation in 200 milliliter of n-decane. The undissolved material wassettled, air dried, and weighed, based on which it was determined thatonly 2% of the clioquinol had dissolved in the n-decane. 100 milliliterof the supernatant (light yellow) was agitated in 100 milliliter of PBS,pH 7.4. Next, the phases were allowed to separate. The lower phase (PBS)was collected and filtered to remove the residue which had formed at thephase interface upon extraction with the organic solvent. Theconcentration of clioquinol in the PBS was determined to be 800nanomolar. This number was arrived at based on two assumptions: (1) 2%of the clioquinol was dissolved in the n-decane; and (2) thepartitioning coefficient is 1/1750 with PBS at 1:1 mixture of n-decaneto clioquinol.

[0367] Resolubilization of in vitro Metal-induced Aβ Aggregates

[0368] First, in order to appraise the efficacy of clioquinol inresolubilizing Aβ aggregates, its ability to resolubilize Aβ aggregatesformed in vitro by the action of Cu²⁺ or Zn²⁺ upon Aβ₁₋₄₀ was examinedFIG. 23 shows resolubilization of metal-induced Aβ aggregate bychelators. Aβ (10 ng/well in buffered saline) aggregation was induced byaddition of metals (5 μM) or acidic conditions (pH 5.5). Aggregates weretransferred to a 0.2 μ nylon membrane by filtration. The aggregates werethen washed (200 μl/well) with TBS alone, TBS containing 2 μM EDTA orTBS with 2 μM clioquinol. The membrane was then fixed, probed withanti-Aβ monoclonal antibody 6E10 and developed for exposure to ECL-film.FIG. 23 shows the relative signal as determined by densitometricanalysis of the ECL-film, calibrated against known amounts of thepeptide. Values are expressed as a % of Aβ signal remaining on thefilter after washing with TBS alone. Clioquinol is hydrophobic, so thatthe reagent must first be solubilized in an organic solvent, and thenpartitioned into the aqueous buffer according to established protocols.

[0369] Like EDTA (FIG. 17), clioquinol significantly resolubilizedprecipitated Aβ. Cu²⁺ partially precipitates Aβ₁₋₄₀ (Bush, A. I., etal., Science 268:1921 (1995)) at pH 7.4. EDTA (2 μM) resolubilized 35%of a Zn²⁺-induced Aβ precipitate, 60% of a Cu²⁺-induced precipitate, and15% of a pH 5.5-induced precipitate. In contrast, clioquinol (2 μM) wasmore effective at resolubilizing the Zn²⁺- and Cu²⁺-induced Aβprecipitates (50%, and 85%, respectively), but was also ineffective atresolubilizing the pH 5.5 precipitate (10%). Since the aggregate at pH5.5 is predominantly β-shect (Wood, S. J. et al., J. Mol Bio.,256:870-877 (1996)), these data indicate that the resolubilization of Aβby clioquinol/EDTA is likely to be due to specific chelation effects.

[0370] Extraction of Agfrom Samples of AD-affected Brains

[0371] Next the ability of clioquinol to extract Aβ deposits from humanbrain was examined. It was found that, like other zinc chelators,clioquinol efficiently increases the resolubilization of AD, compared tothe amount of Aβ resolubilized from the pellet fraction of brainhomogenate by PBS alone. FIG. 24 shows the effect of clioquinol upon theextraction of Aβ from AD-affected brain. Fragments of prefrontal cortexfrom individual post-mortem samples with the histopathological diagnosisof AD were homogenized in PBS, pH 7.4, and then pelleted aftercentrifugation. The pellets were then washed with agitation twice for 30minutes, 4° C., with PBS or PBS containing clioquinol (100% =0.8 μMclioquinol). The suspension was then pelleted (10,000 g for 30 minutes)and the supernatant removed (S1) for western blot analysis usingAβ-specific antibodies (illustrated). The pellet was treated a secondtime in this experiment with agitation and centrifugation, and thesecond supernatant (S2) analysed. The data show typical results bywestern blot.

[0372] In agreement with earlier findings which showed that the optimalconcentration of chelator for the extraction of Aβ is idiosyncracticfrom case to case, and that there is a paradoxical diminution of Aβextraction when the chelator concentration rises above the optimum, itwas found that optimal clioquinol concentrations for Aβ resolubilizationvary in a similar manner (e.g., Specimen #1=0.08 μM, #2=0.8 μM). It wasalso observed that apparently dimeric Aβ was more frequently observed onSDS-PAGE (illustrated), and that in these cases (e.g., Specimen #2) thefirst wash did not resolubilize much Aβ, but the second wash was veryefficient at resolubilizing the peptide. It was surmised that the pelletmass may be coated with adventitial, non-Aβ, proteins that are removedby the first wash, allowing the second treatment access to the Aβcollection. Indeed, further studies have shown that both sustained (for16 hours) and repeated exposure to the chelator increases theresolubilization of Aβ significantly.

[0373]FIG. 25A and 25B show the western blot and accompanyingdensitometric analysis of reolubilization of Aβ from AD-affected brain.FIG. 25A is a western blot showing the effect of clioquinol upon theresolubilization of Aβ from AD-affected brain. In this study, the brainspecimen (from a different case than that of FIG. 24) was homogenizedaccording to the protocols in FIG. 19. In this case a dose-dependentresponse to clioquinol was observed. Synthetic peptide standards thatwere used to calibrate densitometric quantification are shown in the tworight-most lanes.

[0374]FIG. 25B is a chart showing densitometry performed upon theresults in FIG. 25A, above. Proportional change in the amount of Aβrecovered in the extraction of Aβ by clioquinol from human brain isshown. As little as a 1% dilution of clioquinol in PBS (100% =0.8 μM) or8 nM clioquinol is capable of doubling the recovery of Aβ in the solublephase.

[0375] In sequential extraction experiments, as described above,clioquinol (1.12 μM) has been shown to result in a 2.5 fold increase insolubilization of Aβ relative to PBS alone (see FIGS. 25A and 25B).Significantly, the findings the present invention show that very low (8nM) concentrations of clioquinol may resolubilize more than twice theamount of Aβ compared to PBS buffer alone (see FIGS. 25A and 25B). Thissuggests that such low concentrations may be therapeutically effectivein treating amyloidosis, preferrably that occurring in AD-affected humansubjects.

Example 7 Potentiation of Resolubilization of Amyloidfrom AD-affectedBrain Tissue

[0376] Aβ was extracted from cortical tissue obtained from threesubjects with clinically and histopathologically confirmed Alzheimer'sdisease in the presence of 1.6 μM clioquinol (CQ), 2 mM bathocuproine(BC), CQ+BC or PBS. Soluble Aβ (ng/g tissue) was determined asdescribed. Total Aβ was determined following formic acid extraction ofotherwise untreated tissue.

[0377]FIG. 26 illustrates the potentiation of Aβ resolubilization usingclioquinol in combination with bathocuproine by graphically showing theproportion of total Aβ extracted. Table 4 below shows the data depictedin FIG. 26 and, in addition, shows each chelator or chelator combinationin PBS buffer. TABLE 4 Potentiation of Chelator-Promoted AβSolubilization BC + CQ + Subject PBS CQ BC CQ + BC CQ + PBS PBS BC + PBS1 0.74 1.85 3.1 5 0.11 2.36 4.26 2 1.8 4.5 7.2 11.2 2.7 5.4 9.4 3 2.33.4 6 12.7 1.1 3.7 10.4

[0378] The effect of clioquinol and bathocuproine combined is seen to bemuch more than additive. In subject 3, for example, the potentiatedeffect was over twice that of a simple additive effect (10.4 compared to1.1+3.7 or 4.8). These data suggest that combinations of clioquinol andbathocuproine may be particularly effective therapeutic combinations forthe treatment of amyloidosis, in particular, the pathologicalAβ-aggregation manifest in brains of those afflicted with Alzheimer'sdisease.

Example 8 Differential Effects of Chelation of Cerebral Aβ Deposits inAD-affected Subjects Versus Age-matched Controls and the Effect ofMagnesium

[0379] Experiments involving extraction of cerebral tissue fromAD-affected subjects and non-AD, age-matched controls by chelationindicate different resolubilization responses of amyloid depositsbetween the two sample groups with regard to extraction by specificchelators.

[0380] Higher concentrations of chelators with relatively broadspecificity (e.g. EGTA) result in less resolubilization of Aβ deposits.Experiments show that chelation of magnesium negatively affectsresolubilzation of Aβ deposits.

[0381] Materials and Methods

[0382] Cortical tissue was dissected from the frontal poles of frozen ADand age-matched normal brains for which histopathological and clinicaldocumentation were provided. AD tissue was selected according to CERADcriteria (Mirra et al., Neurology 41:479-486 (1991)) with particularattention paid to the presence of neuritic plaques and neurofibrillarytangles. Histological examination of Aβ levels in normal specimensranged from immunohistochemically undetectable to substantially presentin the form of diffuse plaques.

[0383] Suitable quantities of gray matter from each subject were mincedto serve as pools of homogenous tissue. Equal portions (0.5 g unlessotherwise specified) were homogenized (Ika Ultaturax T-25, Janke andKunkel, Staufen, Germany) for 3×30s periods at full speed with a 30second rest between runs in 3 ml of ice-cold phosphate-buffered saline(PBS pH 7.4) containing a cocktail of protease inhibitors (Biorad,Hercules, California.—Note: EDTA was not included in the proteaseinhibitor mixture) or in the presence of chelators or metal ionsprepared in PBS. To obtain the soluble fraction, the homogenates werecentrifuged at 100,000× g for 30 min (Beckman J180, Beckman instruments,Fullerton, California) and the supernatant collected in 1 ml aliquotsand stored on ice or immediately frozen at −70° C. In each experiment,all protein was precipitated from 1 ml of supernatant from eachtreatment group using 1:5 ice cold 10% trichloracetic acid and pelletedin a bench top microfuge (Heraeus, Osteroder, Germany) at 10,000× g. Theremaining pellet was frozen at −70° C.

[0384] The efficiency of the precipitation was validated by applying thetechnique to a sample of whole human serum, diluted 1:10, to which hadbeen added 2 μg of synthetic Aβ₁₋₄₀ or Aβ₁₋₄₂ (W. Keck Laboratory, YaleUniversity New Haven, Conn.). Protein in the TCA pellet was estimatedusing the Pierce BCA kit (Pierce, Rockford, Ill.). The total Aβ load ofunextracted cortex was obtained by dissolving 0.5 g of grey matter in 2ml of 90% formic acid, followed by vacuum drying and neutralization with30% ammonia.

[0385] Precipitated protein was subjected to SDS polyacrylamide gelelectrophoresis (SDS-PAGE) on Novex pre-cast 10-20% Tris-Tricine gelsfollowed by Western transfer onto 0.2 μm nitrocellulose membrane(Biorad, Hercules, Calif.). Aβ was detected using the WO2, G210 or G211monoclonal antibodies (Ida, N., et al., J. Biol. Chem., 271:22908(1996)) in combination with HRP-conjugated rabbit anti-mouse IgG (Dako,Denmark), and visualized using chemiluminescence (ECL, Amersham LifeScience, Little Chalfont, Buckinghamshire, UK). Each gel included two ormore lanes containing known quantities of synthetic Aβ which served asinternal reference standards. Blot images were captured by a Relisysscanner with transparency adapter (Teco Information Systems, Taiwan,ROC) and densitometry conducted using the NIH Image 1.6 program(National Institutes for Health, USA. Modified for PC by ScionCorporation, Frederick, Md.), calibrated using a step diffusion chart.For quantitation of Aβ in brain extracts, the internal referencestandards of synthetic Aβ were utilized to produce standard curves fromwhich values were interpolated.

[0386] In the experiments corresponding to the results shown in FIG. 27,duplicate 0.2 g samples of AD cortical tissue were homogenized andsubjected to ultracentrifugation as described, but using either 1 ml or2 ml of extraction buffer (PBS). Protein was precipitated from theentire supernatant and redissolved in 100 μl of sample buffer. Equalvolumes of TCA-precipitated protein were subjected to Tris-TricineSDS-PAGE and Aβ was visualized as described above.

[0387] In the experiments corresponding to the results shown in FIG.28A, 0.2 g specimens of frontal cortex from AD brain were homogenized inthe presence of 2 ml of PBS or varying concentrations of Cu²⁺ (Cu(SO₄)₂)or Zn²⁺ (Zn(SO₄)₂). Aβ in the high speed supernatant was visualized asdescribed above.

[0388] In the experiments corresponding to the results shown in FIG.28B, 0.2 g specimens of frontal cortex from AD brain were homogenized inthe presence of 2 ml or PBS or 2 mM EGTA. The homogenates were spun at100,000× g for 30 min and the supernatant discarded. The remaining(metal depleted) pellets were rehomogenized in a further 2 ml of eitherPBS alone EGTA alone, 2 mM Mg²⁺ (Ng(Cl)₂.6H₂O) in PBS or 2 mM Ca²⁺(CaCl₂.2H₂O) in PBS and the homogenate subjected to ultracentrifugation.Aβ in the soluble fraction was visualized as described above.

[0389] In the experiments corresponding to the results shown in FIGS.29A and 29B, frontal cortex from AD (n=6) and age-matched,amyloid-positive (n=5) subjects were treated with PBS, TPEN, EGTA or BC(0.1 mM and 2 mM) and soluble Aβ assessed as described above.

[0390] In the experiments corresponding to the results shown in FIG. 30,representative AD (left panels) and aged-matched control specimens(right panels) were prepared as described in PBS or 5 mM BC. Identicalgels were run and Western blots were probed with mAbs WO2 (raisedagainst residues 5-16, recognizes Aβ₁₋₄₀ and Aβ₁₋₄₂), G210 (raisedagainst residues 35-40, recognizes Aβ₁₋₄₀), or G211 (raised againstresidues 35-42, recognizes Aβ₁₋₄₂) (See Ida, N., et al., J. Biol. Chem.,271:22908 (1996)).

[0391] Results and Discussion

[0392] To further explore the involvement of metal ions in thedeposition and architecture of amyloid deposits, the inventors extractedbrain tissue from histologically-confirmed AD-affected subjects and fromsubjects that were age-matched to AD-affected subjects but were notclinically demented (age-matched controls, “AC”) in the presence of avariety of chelating agents and metals. Chelators were selected whichdisplayed high respective affinities for zinc and/or copper relative tomore abundant metal ions such as calcium and magnesium. See Table 5below. TABLE 5 Stability constants of metal chelators Ca Cu Mg Fe Zn AlCo EGTA 10.86 17.57 5.28 11.8 12.6 13.9 12.35 TPEN 3 20.2 n/a 14.4 15.4n/a n/a BC n/a Cu²⁺ n/a n/a  4.1 n/a 4.2 6.1 Cu⁺ 19.1

[0393] logK10 where K=[Metal.Ligand]/[Metal][Ligand]. From: NISTdatabase of critically selected stability constants for metal complexesVersion 2.0 1995.

[0394] A series of titration curves were prepared to determine thechelator concentration at which maximal response was obtained. In theseexperiments, selected chelators were limited to EGTA, TPEN and BC. FIGS.19A-C show that chelators affect the solubilization of Aβ in adose-dependent manner.

[0395] It was found that EGTA and TPEN elicited a significantenhancement in solubilization of Aβ in a pattern of response typified bypeak values at or near 0.004 mM and 0.1 mM, and lower values atconcentrations in between. Both chelators were increasingly ineffectiveat concentrations over 1 mM, and at 2 mM, EGTA virtually abolished thesignal for Aβ. In contrast, BC elicited a typicalconcentration-dependent response with no decline in effectiveness in thelow millmolar range even when extended to 20 mM. Total TCA-precipitatedprotein in the supernatant was assayed and found to be unaffected byeither chelator kind or concentration.

[0396] Recent findings have demonstrated the presence of neurotoxicdimers in the soluble (Kuo, Y -M., et al., J. Biol. Chem. 271:4077-81(1996)) and insoluble (Roher, A. E., et al., Journal of BiologicalChemistry 271:20631-20635 (1996); Giulian, D. et al., J. Neurosci.,16:6021-6037 (1996)) fractions of Aβ extracts of the brains of ADindividuals. FIG. 21 shows that chelator-promoted solubilization of Aβelicits SDS-resistant dimers. Under the preparation conditions used,SDS-resistant dimers were not generally observed in the extracts withPBS alone. Dimers were found to appear in response to chelator-promotedsolubilization of Aβ however.

[0397] The signal for dimeric Aβ was frequently disproportionate to thatof monomeric Aβ and the ratio varied with both the type andconcentration of chelator used (FIG. 21). In contrast, when syntheticAβ₁₋₄₀ was run under identical conditions, the monomer:dimer ratioreflected a predictable and reproducible concentration-dependentrelationship. These data suggest that the dimers observed in extracts ofhuman brain are predominantly an intermediate structural unit generatedby the dissolution of amyloid, resulting in turn from the sequestrationof metals by chelating agents.

[0398]FIG. 28A shows the effect of metals upon the solubility ofbrain-derived Aβ. Precipitation of Aβ was induced by adding eithercopper or zinc to unchelated extracts. The resulting signal for solubleAβ was attenuated, the threshold concentration being between 20 and 50μM for copper and between 5 and 20 μM for zinc. At concentrationsgreater than 100 μM solubility was abolished. Interestingly, at lowerconcentrations of copper there appears to be a transitional stage whereAβ is present in the dimeric form prior to complete aggregation,mirroring the intermediate stage dimers elicited by chelator-mediatedsolubilization.

[0399] In order to confirm that the chelators were effective atsequestering metals at the concentrations employed in these experiments,ICP was used to determine the residual levels of several metals in thepost-centrifugation pellets retained from the experiment described inFIGS. 19A-19C. Of thesix metals tested, zinc levels were reduced by TPENin a dose dependent manner, whereas EGTA affected calcium and magnesium,particularly at higher concentrations. TABLE 6 Residual Metal Levels inPost-Centrifugation (Extracted) Pellets Mg Al Ca Fe Zn Cu (mg/kg)(mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) TPEN (mM) PBS 202 36 573 411 6013 0.004 147 22 322 317 28 10 0.001 192 34 490 512 42 12 0.04 201 22 956322 22 10 0.1 200 60 708 389 21 12 2.0 200 148 419 376 19 11 5.0 205 16377 307 17 10 EGTA (mM) PBS 223 52 1186 266 45 11 0.004 228 73 795 24753 11 0.001 237 43 862 281 49 12 0.04 247 104 1402 438 71 13 0.01 213 61675 272 54 13 2.0 191 62 519 238 27 13 5.0 168 27 455 230 18 12 BC (mM)0.004 234 33 489 231 47 12 0.001 225 88 1306 275 47 13 0.04 226 38 753248 56 15 0.01 223 73 762 256 49 13 2.0 254 42 1602 271 49 14 5.0 238 38912 249 53 15

[0400] Metal levels were measured in 10 AD specimens treated with 0.1 mMTPEN. See Table 7 below. The observed increase in extractable Aβcorrelated with significant depletion in zinc in every case and to alesser extent, copper, when compared with PBS-treated tissue. No othermetal tested was significantly influenced by treatment at thisconcentration. TABLE 7 Residual Metal Levels (Based on 10 AD Specimens)Zn Cu Fe Ca Mg Al PBS 50.7 11.9 227 202 197 44 (+/−SEM) (4.9) (1.5)(28.8) (28.3) (39.1) (46.2) TPEN 33.2 9.8 239 210 230 65 (+/−SEM) (4.1)(1.7) (31.7) (37.0) (39.2) (45.0)

[0401] Given the precipitous decline in extractable Aβ observed whenemploying high concentrations of TPEN or EGTA (see FIG. 9), it washypothesized that magnesium or calcium might also have a significantrole in the Aβ solubility equilibrium. Magnesium or calcium added to thehomogenization buffer produced no appreciable alteration in soluble Aβ.However, using an extract previously depleted of metals by high levelsof EGTA, the addition of magnesium, and to a much lesser extent calcium,led to resolubilization of the precipitated Aβ. FIG. 28B shows that Aβsolubility in metal-depleted tissue samples is restored by supplementingwith magnesium.

[0402] Mindful of the high variability observed between individualsubjects, 6 AD and 5 aged-matched control brains were chosen at randomto determine if the observed phenomena were broadly applicable. Thesespecimens were subjected to chelation treatment at selectedconcentrations of 0.1 or 2.0 mM or with PBS alone. FIG. 29A shows thatpatterns of chelator-promoted solubilization of Aβ differ in AD andaged, non-AD tissue. The chelator-promoted solubilization of Aβ from ADbrains represented an increase of up to 7-fold over that seen with PBSalone; the mean increase for BC being around 4 fold, and that for TPENaround 2 fold. Treatment with EGTA at 2 mM always produced a diminutionin Aβ signal below that observed for the PBS control (See FIG. 29B).

[0403] The effects observed with non-demented, aged-matched controlswere similar with respect to EGTA and TPEN. However, it is noteworthythat the effect of BC was much reduced. In some cases (FIG. 29A, lowerpanel), BC treatment caused an attenuation in soluble Aβ suggesting thatthe amyloid deposits in AD-affected brain respond to this chelator in adifferent fashion than the deposits predominating in non-dementedelderly brain.

[0404] For each subject in the experiments of FIGS. 29A and 29B, theextractable Aβ was derived and calculated as a proportion of the totalpre-extraction Aβ load See Table 8 and 9 below. TABLE 8 AD-AffectedTissue AD 1 2 3 4 5 6 X +/−SEM X C/PBS Total Aβ(μg/g) 10.8 77.0 80.3 6.014.4 16.8 43.0 14.1 PBS μg/g 0.74 1.39 1.04 0.07 3.0 0.06 1.05 0.44 (%of total) (0.1) (1.8) (1.3) (1.1) (2.1) (0.4) (1.2) (0.3) TPEN 2 mM μg/g0.21 3.40 1.80 5.50 5.00 0.28 2.73 0.85 2.60 (% of total) (0.2) (4.4)(2.25) (9.2) (3.5) (1.75) (4.6) (0.9) BC 2 mM μg/g 0.31 5.54 3.62 6.056.03 0.54 4.10 0.86 3.90 (% of total) (0.3) (7.2) (4.5) (10.0) (4.2)(3.4) (5.4) (1.2)

[0405] TABLE 9 Age-Matched Control Tissue AC 1 2 3 4 5 X +/−SEM X C/PBSTotal Aβ(μg/g) 0.7 4.2 2.7 3.2 3.6 2.8 0.60 PBS μg/g 0.17 0.13 0.18 0.100.66 0.25 0.10 (% of total) (25.0) (3.1) (6.7) (3.3) (18.3) (11.3) (4.4)TPEN 2 mM μg/g 0.22 0.38 0.26 0.09 1.06 0.40 0.17 1.6 (% of total)(32.0) (9.0) (9.7) (3.0) (29.5) (16.7) (5.1) BC 2 mM μg/g 0.03 0.24 0.290.08 0.98 0.32 0.16 1.28 (% of total) (5) (5.7) (11.0) (2.6) (27.2)(10.3) (4.6)

[0406] Total Aβ for AD brains ranged from 6-80μg/g wet weight tissue.The percentage of Aβ extractable (one extraction/centrifugationsequence) ranged from 0.33-10%. The corresponding values foraged-matched control brains were 0.68-4.2 μg/g total Aβ and 2.6-29.5%extractable.

[0407] In order to further investigate these different responses tochelators, triplicate blots of AD tissue and control tissue whichdisplayed cerebrovascular and diffuse amyloid deposits were compared.FIG. 30 shows that chelation promotes the solubilization of Aβ₁₋₄₀ andAβ₁₋₄₂ from AD and non-AD tissue. Using 3 different monoclonalantibodies, attempts to detect whether any particular species of Aβ wereselectively affected by chelation were performed. Both Aβ₁₋₄₀ and Aβ₁₋₄₂were liberated by chelation, however the dimeric form of Aβ₁₋₄₀ in bothAD and control tissue predominated. As reported by Roher, A. E., et al.,PNAS 90: 10,836-10,840 (1993), the predominant form of cerebrovascularamyloid is Aβ₁₋₄₂. Somewhat surprisingly, the dimeric form of thishighly aggregating species is absent in the (control) tissue in which itis most favored.

[0408] It has recently been reported that the zinc-dependent InsulinDegrading Enzyme (IDE) has significant Aβ cleavage activity (Perez etal., Proc Soc. for Neuroscience 20: Abstract 321.13 (1997))23. In theexperiments presented here, the disassembly of amyloid is reflected inthe intermediate dimeric species which result from conversion betweensoluble and insoluble forms. Thus, simple inhibition of catalytic enzymeactivity cannot account for the observed increase in soluble Aβ.However, in the event that a proportion of the chelator-mediatedaugmentation of Aβ solubilization was due to inhibition of this enzyme,homogenisations were conducted both in the presence of 1 mM n-ethylamimide (NEM), a potent inhibitor of IDE, and at 37° C. No enhancementof Aβ signal was observed above that of PBS alone for NEM, nor was thereany diminution of signal after incubation at 37° C.

[0409] Discussion

[0410] Metal chelators offer a powerful tool for investigating the roleof metals in the complex environment of the brain, however the strengthsof these compounds may also define their limitations. The broad metalaffinities of most chelators make them rather a blunt instrument.Attempts were made to sharpen the focus of the use of chelators byselecting chelators with a range of affinities for the metals ofinterest. These differences may be exploited by appropriate dilution,thereby favoring the binding of the relatively high affinity ligand(metal for which the chelator has the highest affinity).

[0411] The dilution profiles exhibited by EGTA and TPEN possibly reflecta series of equilibria between different metal ligands and the chelator,whereby the influence of abundant but low affinity metals is observed athigh chelator concentrations and that of the high affinity, but morescarce, metals is predominates at low concentrations of chelator. In thecase of Aβ itself, this explanation is further complicated by thepresence of low and high affinity binding sites for zinc (and copper)(Bush, A. I. et al., J. Biol. Chem., 269:12152-12158 (1994)).

[0412] Bathocuproine with its low affinity for metals other than Cu⁺ iseffective at solubilizing Aβ through a dilution range over 3 orders ofmagnitude, and interestingly, does not diminish in effectiveness at thehighest levels tested. The particular affinity of BC for Cu⁺ has beenexploited to demonstrate that in the process of binding to APP, Cu²⁺ isreduced to Cu⁺ resulting in the liberation of potentially destructivefree radicals (Multhaup, G., et al., Science 271:1406-1409 (1996)). Ithas been shown that Aβ has a similar propensity for reducing copper withconsequent free radical generation (Huang, X., et al., J. Biol. Chem.272:26464-26470 (1997)).

[0413] Although the predicted reduction in copper in extraction pelletstreated with BC has not been demonstrated, it is possible that the ratioof Cu²⁺ to Cu⁺ has been affected. At this stage, however, the means toevaluate the relative contributions of divalent and reduced forms to thetotal copper content of such extraction pellets are not available.

[0414] In addition to their primary metal binding characteristics,chelators are a class of compounds which vary in hydrophobicity andsolubility. Their capacity to infiltrate the highly hydrophobic amyloiddeposits may therefore be an important factor in the disassembly ofaggregated Aβ. It is also possible that the chelators are also acting toliberate intracellular stores of Aβ in vesicular compartments asmetal-bound aggregates. Preliminary data from our laboratory indicatesthat this may be the case with platelets.

[0415] The variability between subjects is consistent, reflecting theheterogeneity of the disease in its clinical and histopathologicalexpression. Despite this, a consistent pattern of response to theactions of chelators by tissue from both AD and non-AD subjects isobserved. This universality of the phenomenon of chelator-mediatedsolubilization is strongly suggestive that metals are also involved inthe assembly of amyloid deposits in normal individuals, although thedissimilar patterns of response suggest that different mechanisms areoperating in the disease and non-pathological states.

[0416] On the basis of the evidence presented here and the in vitrodata, it is proposed that zinc functions in the healthy individual topromote the reversible aggregation of Aβ, counteracted by magnesiumacting to maintain Aβ solubility. Further, the disease state ischaracterized by an unregulated interaction with copper resulting in thegeneration of free radicals.

[0417] A functional homoeostatic mechanism implies equilibrium betweenintracellular copper and zinc (and perhaps other metals) normallypresent in trace amounts, for which Aβ has strong affinity, and moreabundant metals which bind less strongly to Aβ. Zinc is of particularinterest because the anatomical distribution of zinc correlates with thecortical regions most susceptible to amyloid plaque formation (Assaf, S.Y. & Chung, S. H., Nature, 308:734-736 (1984)).

[0418] It has recently been demonstrated (Huang, X., et a., J. Biol.Chem. 272:26464-26470 (1997)) that zinc-promoted aggregation ofsynthetic Aβ is reversible by the application of EDTA. Thetightly-regulated neurocortical zinc transport system might provide aphysiological parallel for this chelator-mediated disaggregation bymoving zinc quickly in and out of the intraneuronal spaces.

[0419] Copper, wvhile binding less avidly to Aβ than zinc (Bush, A. I.,et al., J. Biol. Chem. 269:12152-12158 (1994)) has greater potential toinflict damage via free radical generation, resulting polymers are notreversible (see Example 10, below). Slight alterations in thetransportation and/or metabolism of metals resulting from age-relateddeterioration of cellular processes may provide the environment for arapid escalation of metal-mediated Aβ accretion which eventuallyoverwhelms regulatory and clearance mechanisms. In describing amechanism for Aβ homeostasis this model for amyloid deposition implies apossible physiological role for Aβ whereby aggregation anddisaggregation may be effected through regulation or cortical metallevels and that the predominantly sporadic character of AD reflectsindividual differences in the brain milieu. Such a mechanism by no meansrules out other genetic, environmental, inflammatory or other processesinfluencing the progression of the disease. Furthermore, indemonstrating the effectiveness of chelators in solubilising amyloid, itis suggested herein that agents of this type are useful for therapeuticor prophylactic use in AD.

Example 10 Formation of SDS-resistant Aβ Polymers

[0420] The cause for the permanent deposition of Aβ in states such asAlzheimer's Disease (AD) and Down's Syndrome (DS) are unknown, but theextraction of Aβ from the brains of AD and DS patients indicates thatthere are forms of Aβ that can be resolubilized in water and run as amonomer on SDS-PAGE (Kuo, Y -M., et al., J. Biol. Chem. 271:4077-4081(1996); see also Example 9 above), and forms that manifest SDS-, urea-and formic acid-resistant polymers on PAGE (Masters, C. L. et al., Proc.Natl. 4cad. Sci. USA 82:4245-4249 (1985); Dyrks, T., et al., J. Biol.Chem. 267:18210-18217 (1992); Roher, A. E., et al., Journal ofBiological Chemistry 271:20631-20635 (1996). Thus, the extraction ofSDS-resistant Aβ polymers from plaques implicates polymerization as apathogenic mechanism that promotes the formation of AD amyloid.

[0421] The exact mechanism underlying the formation of SDS-resistantpolymeric Aβ species remains unresolved. Recently, we found that Aβreduces both Cu²⁺ and Fe³⁺ (Huang, X., et al., J. Biol. Chem.272:26464-26470 (1997)), providing a mechanism whereby a highly reactivespecies could promote the modification of proteins via an oxidativemechanism. In this study we test the ability of Cu²⁺ and Fe³⁺ to promoteSDS-resistant Aβ polymerization.

[0422] Materials and Methods

[0423] Human Aβ₁₋₄₀ peptide was synthesized, purified and characterizedas described above. Rat Aβ₁₋₄₀ was obtained from Quality ControlBiochemicals, Inc. (Hopkinton, Mass.). Peptides were analyzed and stocksolutions prepared as described above.

[0424] As above, electronic images captured using the Fluoro-S ImageAnalysis System (Bio-Rad, Hercules, Calif.) were analyzed usingMulti-Analyst Software (Bio-Rad, Hercules, Calif.). Thischemiluminescent image analysis system is linear over 2 orders ofmagnitude and has comparable sensitivity to film.

[0425] Human AD derived SDS-resistant polymers were solublized in formicacid, and then dialyzed with 5 changes of 100 mM ammonium bicarbonate,pH 7.5. The solublized peptide was then used for subsequent chelationexperiments.

[0426] Results and Discussion

[0427] The generation of SDS-resistant Aβ polymers by metal ions wastested by incubating Cu²⁺ (30 μM) or Zn²⁺ (30 μM) at pH 6.6, 7.4 and 9.0with Aβ₁₋₄₀. As shown in FIG. 9, Western blot analysis of samplesincubated with Cu²⁺ and run under SDS denaturing and P-mercaptoethanolreducing conditions revealed an increase in dimeric, trimeric and higheroligomeric Aβ species over time. The dimer and trimer had molecularweights of approximately 8.5 kD and 13.0 kD, respectively. Imageanalysis indicated 42% and 9% conversion of the monomer to dimer andtrimer, respectively, in samples incubated at pH 7.4 after 5 d. Theconversion of monomer to the dimer and trimer was 29% and 2%,respectively, at pH 6.6 after 5 d.

[0428] In contrast, changes in [H+] alone did not induce SDS-resistantAβ₁₋₄₀ polymerization. Less than 4% of the peptide was converted to theSDS-resistant dimer after 5 d in samples incubated at pH 6.6, 7.4 or9.0, most likely as a result of contaminating Cu²⁺ in the buffer and Aβsolutions. Cu²⁺ contamination of chelex-treated PBS was up to 0.5,μM asdetermined by ion coupled plasma-atomic emission spectroscopy (ICP-AES).Although Zn²⁺ induces rapid aggregation of Aβ₁₋₄₀ (Bush, A. I., et al.,J. Biol. Chem. 268:16109 (1993); Bush, A. I., et al., J. Biol. Chem.269:12152 (1994); Bush, A. I., et al., Science 265:1464-1467 (1994);Bush, A. I., et al., Science 268:1921-1922 (1995); Atwood et al.,submitted; Huang, X. et al., J. Biol. Chem. 272:26464-26470 (1997)), itdid not induce SDS-resistant Aβ polymerization (FIG. 9) as previouslyreported (Bush, A. I., et al., Science 268:1921-1922 (1995)).

[0429] Aβ₁₋₄₂ is the predominant species found in amyloid plaques(Masters, C. L. et al., Proc. Natl. Acad. Sci. USA 82: 4245 (1985);Murphy, G. M., et al., Am. J. Pathol. 144:1082-1088 (1994); Mak, K., etal., Brain Res. 667:138-142 (1994); Iwatsubo, T., et al., Ann. Neurol.37:294-299 (1995); Mann et al., Ann. Neurol. 40:149-156 (1996)).Therefore, the ability of Aβ₁₋₄₀ and Aβ₁₋₄₂ to form SDS-resistantpolymers was compared.

[0430] In contrast to Cu²⁺-induced SDS-resistant Aβ₁₋₄₀ polymerizationover days, SDS-resisitant Aβ₁₋₄₂ polymerization occurred within minutesin the presence of Cu²⁺ (FIG. 31A). Unlike Aβ₁₋₄₀ where Cu²⁺ induces theformation of a SDS-resistant dimeric species first, Aβ₁₋₄₂ initiallyforms an apparent trimer species in the presence of Cu²⁺. Over time,dimeric and higher polymeric species also appear in Aβ₁₋₄₂ incubationswith Cu²⁺ at both pH 7.4 and 6.6. The greater Cu²⁺ induced Add ₄₂polymerization observed at pH 6.6 compared with pH 7.4 in samplesincubated for 30 min. was reversed after 5 d. At pH 6.6, both Aβ₁₋₄₀ andAβ₁₋₄₂ exist in an aggregated form within minutes. Therefore, theformation of these polymeric species occurs within Aβ aggregates and theformation of SDS-resistant Aβ polymers is independent of aggregationstate (see below). Similar results were obtained using the monoclonalantibody 4G8.

[0431] Since redox active Fe (Smith, M. A., et al., Proc. Natl. Acad.Sci. USA 94:9866 (1997)) and ferritin (Grudke-Iqbal, I., et al., ActaNeuropathol. 81:105 (1990)) are found in amyloid lesions, experimentswere performed to determine if Fe could induce SDS-resistantpolymerization of Aβ₁₋₄₀ and Aβ₁₋₄₂ (FIG. 31A). Fe³⁺ did not induceAβ₁₋₄₀ polymerization above background levels with either peptide. Thesmall increase in polymeric Aβ₁₋₄₀ and Aβ₁₋₄₀ in samples with no metalions reflects a small contaminating concentration of Cu²⁺.

[0432] The formation of amyloid plaques is not a feature of aged rats(Johnstone, E. M., et al., Mol. Brain Res. 10:229 (1991); Shivers etal., EMBO J., 7:1365-1370 (1988)). To test whether rat Aβ₁₋₄₀ would formSDS-resistant Aβ polymers, rat Aβ₁₋₄₀ was incubated with Cu²⁺ and Fe³⁺at pH 7.4 and 6.6 (FIG. 31B). Neither metal ion induced SDS-resistant Aβpolymers (Huang, X. et al., J. Biol. Chem. 272:26464-26470 (1997)). Thebinding and reduction of Cu²⁺ by rat Aβ₁₋₄₀ is markedly decreasedcompared to that of human Aβ₁₋₄₀ (Huang, X. et al., J. Biol. Chem.272:26464-26470 (1997)). This result suggests that the generation ofSDS-resistant Aβ polymers is dependent upon the binding and reduction ofCu²⁺ by Aβ.

[0433] Tests were performed to determine the concentration of Cu²⁺required to induce the formation of SDS-resistant Aβ₁₋₄₀ and Aβ₁₋₄₂polymers. Aβ₁₋₄₀ and Aβ₁₋₄₂ were incubated with different [Cu²⁺] (0-30μM) at pH 7.4 and 6.6 and the samples analyzed by Western blot and thesignal quantitated using the Fluoro-S Image Analysis System (Bio-Rad,Hercules, Calif.) as previously described.

[0434] At pH 7.4, the increase in polymerization was barely detectableas [Cu²⁺] was increased from 0.5 to 1 μM, but under mildly acidicconditions (pH 6.6), SOS-resistant polymerization could be detected(over 3-fold increase in dimerization (Table 10A). TABLE 10A Cu²⁺ -Induced SDS-Resistant Polymers of Aβ₁₋₄₀ [Cu²⁺] Monomer Dimer TrimerTetramer Pentamer pH 7.4 0 96.8 3.2 <0.1 0 0 0.5 94.8 4.9 0.3 0 0 1 93.65.9 0.6 0 0 5 84.3 14.2 1.5 0 0 10 85.2 13.2 1.6 0 0 30 76.2 19.1 4.7 00 pH 6.6 0 97.9 2.1 <0.1 0 0 0.5 97.6 2.2 0.2 0 0 1 92.6 7.3 0.1 0 0 590.1 9.8 0.1 0 0 10 79.4 16.1 4.5 0 0 30 74.5 13.2 12.2 0 0

[0435] A similar Cu²⁺ concentration and pH dependent increase inSDS-resistant Aβ₁₋₄₂ polymers also was observed (Table 10B), butSDS-resistant polymerization occurred at much lower [Cu⁺]. TABLE 10BCu²⁺—Induced SDS-Resistant Polymers of Aβ₁₋₄₂ [Cu²⁺] Monomer DimerTrimer Tetramer Pentamer pH 7.4 0 76.61 0 16.0 5.5 1.9 0.5 70.7 0 20.56.2 2.5 1 64.9 0 23.6 7.4 4.0 5 56.1 0 31.8 8.7 4.1 10 55.1 0 30.3 10.34.3 30 57.1 0 31.1 8.3 4.2 pH 6.6 0 61.0 0 27.3 8.6 3.8 0.5 52.1 0 33.812.0 3.0 5 59.6 0 30.0 7.1 3.2 10 52.3 0 31.7 13.6 2.2

[0436] Aβ₁₋₄₀ polymerization was not detected with increasing Fe³⁺concentrations at any pH. Therefore, of the metal ions known to interactwith Aβ, only Cu²⁺, whose ability to aggregate and bind Cu²⁺ undermildly acidic conditions is enhanced, is capable of inducingSDS-resistant Aβ polymerization.

[0437] Oxygen radical mediated chemical attack has been correlated withan increase in protein and free carbonyls (Smith, C. D., et a., Proc.Natl. Acad. Sci. USA 88:10540 (1991); Hensley, K., et al., J. Neurochem.65:2146 (1995); Smith, M. A., et al., Nature 382:120 (1996)) andperoxynitrite-mediated protein nitration (Good, P. F., et al., Am. J.Pathol. 149:21 (1996); Smith, M. A., et al., Proc. Natl. Acad. Sci. USA94:9866 (1997)).

[0438] Aβ is capable of reducing Cu²⁺ and H₂O₂ is produced in solutionscontaining Aβ and Cu²⁺ or Fe³⁺ (Huang, X. et al., i J. Biol. Chem.272:26464-26470 (1997)). As shown above, the generation of SDS-resistantAβ polymers in the order Aβ₁₋₄₂>>Aβ₁₋₄₀>>rat Aβ₁₋₄₀ in the presence ofCu²⁺ correlates well with the generation of Cu⁺ and reactive oxygenspecies (ROS; OH⁻, H₂O₂ and O₂ ⁻: Huang, X. et al., J. Biol. Chem.272:26464-26470 (1997)) by each peptide.

[0439] The increased generation of SDS-resistant Aβ polymers in thepresence of Cu²⁺ compared to Fe³⁺ also was correlated with thegeneration of the reduced metal ions, respectively (Huang, X. et al., J.Biol. Chem. 272:26464-26470 (1997)). The increase in SDS-resistant Aβpolymerization seen under mildly acidic conditions may be a result ofthe higher [H⁺] driving the production of H₂O₂ dismutated from O₂ ⁻ withthe subsequent generation of OH. via Fenton-like chemistry inducing amodification of Aβ that results in SDS-resistant Aβ polymers (see FIG.12, showing a schematic of the proposed mechanism of Aβ-mediated reducedmetal/ROS production).

[0440] To confirm whether ROS were involved in the generation ofSDS-resistant polymers, experiments were performed to determine whetherCu in the presence or absence of H₂O₂ could promote Aβ polymerization(FIG. 32A). A similar level of Aβ₁₋₄₂ polymerization was observed in thepresence of Cu²⁺ or Cu⁺, indicating that the reduced metal ion alone wasnot capable of increasing Aβ polymerization. Likewise, polymerization ofAβ₁₋₄₂ in the presence of H₂O₂ was low and equivalent to control levels.However, the addition of Cu²⁺ or Cu⁺ to Aβ in the presence of H₂O₂induced a similar, marked increase in dimers, trimers and tetramerswithin 1 hour. After 1 day, higher molecular weight polymers (>18 kD)were generated (from the oligomers), with a subsequent reduction in thelevels of monomer, dimer, trimer and tetramer only with the coincubationof H₂O₂ and Cu²⁺.

[0441] Both the reduced and oxidized forms of Cu produced similar levelsof polymerization in the presence of H₂O₂. In contrast, Fe³⁺ of Fe²⁺ didnot induce as much polymerization as Cu²⁺ in the presence of H₂O₂ after1 day incubation (FIGS. 32A and 32B). Since Fe³⁺ is not reduced asefficiently as Cu²⁺ by Aβ (Huang, X., et al., J. Biol. Chem.,272:26464-26470 (1997)), and Cu⁺ is rapidly converted to Cu²⁺ insolution, these results suggest that the reduction reaction is requiredfor the polymerization reaction to proceed.

[0442] It was confirmed that the reduction of Cu²⁺ was required forgenerating SDS-resistant Aβ polymerization by incubating Aβ₁₋₄₂ and Cu²⁺with and without bathocupoinedisulfonic acid (BC), a Cu⁺ specificchelator (FIG. 32C). There was a marked decrease in polymerization,indicating that Cu⁺ generation was crucial for the polymerization of Aβ.It is possible that the decreased polymerization may be due to chelationof Cu²⁺ by BC, however given the low binding affinity of BC for Cu²⁺compared with Aβ, it seems likely that the chelation of Cu⁺ by BCprevents it from inducing SDS-resistant Ad polymerization. Therefore, Aβmay undergo a hydroxyl radical modification that promotes its assemblyinto SDS-resistant polymers.

[0443] If H₂O₂ is required for the polymerization reaction underphysiological conditions, the removal of H₂O₂ and it's precursors O₂ andO₂ ⁻ (Huang, X., et al., J. Biol. Chem., 272:26464-26470 (1997)) shoulddecrease SDS-resistant polymerization. To confirm that H₂O₂ generated inthe presence of Aβ and Cu²⁺ was required for the polymerizationreaction, Aβ₁₋₄₂ was incubated with or without Cu²⁺ in the presence ofTCEP (FIG. 33A). TCEP significantly reduced the level of polymerizationin samples with and without Cu²⁺ over 3 days. This indicates that thegeneration of H₂O₂ is required for the polymerization of Aβ.

[0444] To confirm that the generation of O₂ ⁻ was required forSDS-resistant Aβ polymerization, Aβ₁₋₄₂ was incubated with and withoutCu²⁺ at pH 7.4 and 6.6 under argon in order to decrease the reduction ofmolecular O₂ (FIG. 33B). Argon-purging of the solution markedlydecreased Aβ₁₋₄₂ polymerization under each condition, indicating thatthe generation of ROS is required for the polymerization of Aβ.

[0445] Taken together, these results indicate that polymerization occursas a result of Haber-Weiss chemistry where the continual reduction ofCu²⁺ by Aβ provides a species for the reduction of molecular O₂ and thesubsequent generation of O₂ ⁻, H₂O₂ and OH. The binding and reduction ofCu²⁺ by Aβ is supported by the finding that the incubation of Fe³⁺, H₂O₂and ascorbic acid with Aβ₁₋₄₀ (FIG. 33A) and Aβ₁₋₄₂ does not induceSDS-resistant polymerization equivalent to Cu²⁺ with H₂O₂ alone. Sinceascorbic acid effectively reduces Fe³⁺, the reduction of a metal ionthat is not bound to Aβ is insufficient to induce significantSDS-resistant polymerization.

[0446] The formation of SDS-resistant polymers of Aβ by thismetal-catalyzed oxidative mechanism strongly suggested that a chemicalmodification to the peptide backbone allows the formation of the polymerspecies. To test if the SDS-resistant polymers were covalently linked,SDS-resistant polymers generated by incubating Aβ₁₋₄₂ with Cu²⁺ at pH 74 and 6.6, or Aβ₁₋₄₂ with Cu²⁺ plus H₂O₂ were subjected to treatmentwith urea (FIG. 34A) and guanidine HCl, chaotrophic agents known todisrupt H-bonding. Urea and guanidine HCl did not disrupt theSDS-resistant polymers at 4.5 M, and only slightly at 9M, suggestingthat the SDS-resistant polymers were held together by high-affinitybonds, but not hydrogen bonding alone. HPLC-MS analyses confirmed nocovalent modification of the peptide and no evidence of intermolecularcovalent crosslinking.

[0447] Since covalent and/or hydrogen bonding were not involved inpolymer formation, experiments were performed to detemine whether Cu²⁺coordination of the complex by ionic interactions was allowing for theformation of the SDS-resistant polymer species. To disrupt these ionicinteractions, different chelating agents were added to a solutioncontaining Cu²⁺-induced Aβ₁₋₄₀ or Aβ₁₋₄₂ SDS-resistant polymersgenerated at pH 7.4 (FIGS. 34B and 34C).

[0448] All chelators significantly reduced the amount of Aβ₁₋₄₀ orAβ₁₋₄₂ SDS-resistant polymers. EDTA was less effective in destabilizingthe polymers, possibly due to its larger molecular mass, and loweraffinity for Cu²⁺. EDTA reduced the amount of Aβ₁₋₄₀ polymers, butincreased the amount of Aβ₁₋₄₀ polymers at pH 7.4. This may be due tothe fact that EDTA can enhance the redox potential of Cu under certainconditions.

[0449] Cu²⁺-induced SDS-resistant polymers generated at pH 6.6 were alsodisrupted with chelation treatment to a similar extent. These resultssuggest that the chelation of Cu²⁺ away from Aβ results in thedisruption of the polymer complex and the release of monomer species.Thus, there is an absolute requirement for metal ions in thestabilization of the SDS-resistant polymer complex.

[0450] The SDS-resistant polymers generated with Cu²⁺ are similar tothose extracted from post-mortem AD brains (Roher, A. E., et al.,Journal of Biological Chemistry 271:20631-20635 (1996)). To determine ifthese human oligomeric Aβ species could be disrupted by metal chelators,TETA and BC were incubated with Aβ oligomers extracted from human brain.FIG. 30E shows that both TETA and BC significantly increased the amountof monomer Aβ in samples treated with these chelators. Although theincrease in the amount of monomer was small, these results suggest thathuman oligomeric Aβ species are partially held together with metal ions.Importantly, this result indicates the potential of chelation therapy asa means of reducing amyloidosis.

[0451] To examine whether conformational changes could disrupt theSDS-resistant polymers, solutions of SDS-resistant Aβ₁₋₄₂ polymers inthe presence or absence of Cu²⁺ were incubated with the α-helicalpromoting solvent system DMSO/HFIP, or under acidic conditions (pH 1)(FIG. 34D). These conditions reduced the amount of polymer compared tountreated controls at both pH 7.4 and 6.6, indicating that an alterationin the conformation of Aβ₁₋₄₂ to the α-helical conformation coulddisrupt the strong Aβ-Cu²⁺ ionic interactions. This provides indirectevidence that the polymer structures are likely to be in the morethermodynamically favorable β-sheet conformation, such as those found inneuritic plaques.

[0452] SDS-resistant Aβ polymers, such as that found in the AD-affectedbrain, are likely to be more resilient to proteolytic degradation andmay explain the permanent deposition of Aβ in amyloid plaques.Incubation of SDS-resistant Aβ polymers with proteinase K resulted incomplete degradation of both monomer and oligomeric Aβ species. Sinceprotease treatment is incapable of digesting hard core amyloid, someform of covalent crosslinking of the peptide following its depositionmay occur over time that prevents proteolytic digestion. This mayexplain the limited disruption of human SDS-resistant Aβ oligomerscompared to the Cu-mediated SDS-resistant polymers generated in vitro.

[0453] Soluble Aβ₁₋₄₀ and Aβ₁₋₄₂ both exist in phosphate buffered salineas non-covalent dimers (Huang, X., et al., J. Biol. Chem.272:26464-26470 (1997); and unpublished observations). Disruption ofionic and hydrogen bonding of Aβ in the soluble and aggregated forms (pHor Zn²⁺) by the ionic detergent SDS results in the complete dissociationof Aβ into the monomer species as detected on SDS-PAGE (FIGS. 9, 28-30).The formation of SDS-resistant polymers of Aβ over time in the presenceof Cu²⁺ (FIGS. 9, 31A-31B, 32C) suggests that conformational orstructural alterations allow for the formation of a thermodynamicallymore stable complex.

[0454] Although no covalent crosslinking between peptides was detected,it is possible that a covalent modification(s) takes place within thepeptide backbone that allows for a high affinity association to formbetween the peptide and Cu²⁺. Thus, a chemical modification to thepeptide may increase the affinity of the polymer for Cu²⁺ and theformation of a stable complex. Alternatively, the requirement formolecular oxygen suggests that Cu may be coordinated by oxygen or ROS inthe formation of SDS-resistant polymers.

[0455] The formation of SDS-resistant polymers was dependent upon thebinding and reduction of Cu²⁺. The binding of Cu²⁺ to Aβ was confirmedby the detection of Cu²⁺ in both the monomer and dimer followingSDS-PAGE. The [Cu²⁺] of PVDF membrane containing the immobilized peptidespecies was measured by ICP-AES (unpublished observations; Huang, X., etal., J. Biol. Chem. 272:26464-26470 (1997)) and correlated with thegeneration of SDS-resistant polymers for each species.

[0456] Cu²⁺ coordination between Aβ molecules was required in order tomaintain the structure since chelation treatment disrupted the in vitrogenerated SDS-resistant polymer (FIGS. 34B-34E). Human SDS-resistant Aβpolymers also were disrupted with the Cu⁺ specific chelator BCindicating Cu coordination in the stabilization of these structures(FIG. 34E). Together with the fact that Cu specific chelators canextract more SDS-resistant Aβ polymers from AD brains in aqueous buffer(see Example 9), these results implicate Cu²⁺ in the generation ofSDS-resistant polymers in vivo.

[0457] Fe³⁺ did not induce the formation of SDS-resistant polymers invitro (FIGS. 31A) as previously reported except in the presence ofexcess H₂O₂ or ascorbic acid as previously reported (Dyrks, T., et al.,J. Biol. Chem. 267:18210-18217 (1992); data not shown). Dyrks, T., etal. did, however observe significant increases in SDS-resistantpolymerization with metal-catalyzed oxidation systems (Fe-hemin,Fe-hemoglobin or Fe-EDTA) in the presence of H₂O₂. The Aβ₁₋₄₂ used intheir experiments was likely to be Cu-bound as it was extracted from awheat germ expression system and already was present as SDS-resistantoligomers. Thus, it is possible that Cu-bound Aβ used in theseexperiments contributed to the increased SDS-resistant polymerizationobserved in the Fe-catalyzed oxidation systems. Although Fe³⁺ is reducedby Aβ (Huang, X., et al., J. Biol. Chem. 272:26464-26470 (1997)), it isunable to effectively coordinate the complex like Cu (FIG. 32B).

[0458] Fe²⁺ is found in much higher concentrations in the brains of ADpatients compared with age-matched controls (Ehmann, W. D., et al.,Neurotoxicol. 7:197-206 (1986); Dedman, D. J., et al., Biochem. J.287:509-514 (1992); Joshi, J. G., et al., Environ. Health Perspect.102:207-213 (1994)). This is partly attributable to the increasedferritin rich microglia and oligodendrocytes that localize to amyloidplaques (Grudke-Iqbal, I., et al., Acta Neuropathol. 81:105 (1990);Conner, J. R., et al., J. Neurosci. Res. 31:75-83 (1992); Sadowki, M.,et al., Alzheimer's Res. 1:71-76 (1995)).

[0459] Recently, redox active Fe was localized to amyloid lesions(Smith, M. A., et al., Proc. Natl. Acad. Sc. USA 94:9866 (1997). WhileFe is normally sequestered by metalloproteins, this localization offerritin-rich cells around amyloid deposits, and the very highconcentrations of iron in amyloid plaques (Conner, J. R., et al., J.Neurosci Res. 31:75-83 (1992); Markesbery, W. R. and EBhmann, W. D.,“Brain trace elements in Alzheimer's disease,” in Terry, R. D., et al.,eds., Alzheimer Disease, Raven Press, New York (1994), pp. 353-368)suggests that reduced Fe released from ferritin and transferrin undermildly acidic conditions could be available for Fenton chemistry and theformation of SDS-resistant polymers. However, even in the presence of aFe-ROS generating system (ascorbic acid, H₂O₂ and Fe) the generation ofSDS-resistant Ad polymers in vitro was low (FIG. 33A) and may beexplained by Cu²⁺ contamination of the buffers.

[0460] Interestingly, diffuse plaques, which may represent the earlieststages of plaque formation, are relatively free of ferritin-rich cells(Ohgami, T., et al., Acta Neuropathol 81:242-247 (1991)). Therefore, theaccretion of iron in amyloid plaques may be a secondary response to theneurodegeneration caused by the reduction of Cu²⁺ and the generation ofROS by Aβ and the formation of neurotoxic SDS-resistant Aβ polymers.

[0461] Structural differences between Aβ₁₋₄₀ and Aβ₁₋₄₂ may allow forthe formation of a thermodynamically stable dimer in the case of Aβ₁₋₄₀and trimer in the case of Aβ₁₋₄₂ (FIGS. 31A, 34B and 34C). Irrespectiveof this, the increased generation of SDS-resistant polymers by Aβ₁₋₄₂compared to Aβ₁₋₄₀ is most likely explained by the increased ability ofAβ₁₋₄₂ to reduce Cu and generate ROS. Since the addition of exogenousH₂O₂ to Aβ₁₋₄₂ increases the generation of dimeric SDS-resistantpolymers of Aβ₁₋₄₂ (FIGS. 32A and 32B), this dimeric species may be anintegral intermediate in the formation of the SDS-resistant Aβ trimers,and may explain why Aβ₁₋₄₀ polymerization occurs more slowly.

[0462] AD Pathology

[0463] The present invention indicates that the manipulation of thebrain biometal environment with specific agents acting directly (e.g.chelators and antioxidants) or indirectly (e.g., by improving cerebralenergy metabolism) provides a means for therapeutic intervention in theprevention and treatment of Alzheimer's disease.

[0464] Those skilled in the art will appreciate that the inventiondescribed herein is susceptible to variations and modifications otherthan those specifically described. It is to be understood that theinvention includes all such variations and modifications. The inventionalso includes all of the steps, features, compositions and compoundsreferred to or indicated in this specification, individually orcollectively, and any and all combinations of any two or more of saidsteps or features.

[0465] All patents and publications cited in the present specificationare incorporated by reference herein in their entirety.

What is claimed is:
 1. A method of treating amyloidosis in a subject,said method comprising administering to said subject a combination of(a) a metal chelator selected from the group consisting of:bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, penicillamine,TETA, and TPEN, or hydrophobic derivatives thereof; and (b) clioquinol,for a time and under conditions to bring about said treatment; whereinsaid combination reduces, inhibits or otherwise interferes withAβ-mediated production of radical oxygen species.
 2. The method of claim1 wherein the metal chelator is bathocuproine.
 3. The method of claim 1further comprising administering a supplement selected from the groupconsisting of: ammonium salt, calcium salt, magnesium salt, and sodiumsalt.
 4. The method of claim 3 wherein the supplement is magnesium salt.5. The method of claim 1 further comprising administering to the subjectan effective amount of a compound selected from the group consisting of:rifampicin, disulfiram, and indomethacin, or a pharmaceuticallyacceptable salt thereof.
 6. A method of treating amyloidosis in asubject, said method comprising administering to said subject aneffective amount of a combination of (a) a salt of a metal chelator,wherein said chelator is selected from the group consisting of:bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, penicillamine,TETA, and TPEN, or hydrophobic derivatives thereof, and (b) clioquinol;wherein said salt of the metal chelator is selected from the groupconsisting of: ammonium, calcium, magnesium, and sodium; and whereinsaid combination reduces, inhibits or otherwise interferes withAβ-mediated production of radical oxygen species.
 7. The method of claim6 wherein the metal chelator is bathocuproine.
 8. The method of claim 6wherein the salt of a metal chelator is a magnesium salt.
 9. The methodof claim 6 further comprising administering to said subject a compoundselected from the group consisting of: rifampicin, disulfiram, andindomethacin, or a pharmaceutically acceptable salt thereof.
 10. Amethod of treating amyloidosis in a subject, said method comprisingadministering to said subject an effective amount of a combination of(a) a chelator specific for copper, and (b) clioquinol; wherein saidcombination reduces, inhibits or otherwise interferes with Aβ-mediatedproduction of radical oxygen species.
 11. The method of claim 10 whereinthe chelator specific for copper is specific for the reduced form ofcopper.
 12. The method of claim 11 wherein the chelator is bathocuproineor a hydrophobic derivative thereof.
 13. A method of treatingamyloidosis in a subject, said method comprising administering to saidsubject an effective amount of a combination of (a) an alkalinizingagent and (b) clioquinol; wherein said combination reduces, inhibits orotherwise interferes with Aβ-mediated production of radical oxygenspecies.
 14. The method of claim 13 wherein the alkalinizing agent ismagnesium citrate.
 15. The method of claim 13 wherein the alkalinizingagent is calcium citrate.
 16. A method of treating amyloidosis in asubject, said method comprising administering to said subject acombination of (a) a metal chelator selected from the group consistingof: bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, penicillamine,TETA, and TPEN, or hydrophobic derivatives thereof; and (b) clioquinol,for a time and under conditions to bring about said treatment; whereinsaid combination prevents formation of Aβ amyloid, promotes, induces orotherwise facilitates resolubilization of Aβ deposits, or both.
 17. Themethod of claim 16 wherein the metal chelator is bathocuproine.
 18. Themethod of claim 16 further comprising administering a supplementselected from the group consisting of: ammonium salt, calcium salt,magnesium salt, and sodium salt.
 19. The method of claim 18 wherein thesupplement is magnesium salt.
 20. The method of claim 16 furthercomprising administering to the subject an effective amount of acompound selected from the group consisting of: rifampicin, disulfiram,and indomethacin, or a pharmaceutically acceptable salt thereof.
 21. Amethod of treating amyloidosis in a subject, said method comprisingadministering to said subject an effective amount of a combination of(a) a salt of a metal chelator, wherein said chelator is selected fromthe group consisting of: bathocuproine, bathophenanthroline, DTPA, EDTA,EGTA, penicillamine, TETA, and TPEN, or hydrophobic derivatives thereof,and (b) clioquinol; wherein said salt of the metal chelator is selectedfrom the group consisting of: ammonium, calcium, magnesium, and sodium;and wherein said combination prevents formation of Aβ amyloid, promotes,induces or otherwise facilitates resolubilization of Aβ deposits, orboth.
 22. The method of claim 21 wherein the metal chelator isbathocuproine.
 23. The method of claim 21 wherein the salt of the metalchelator is a magnesium salt.
 24. The method of claim 21 furthercomprising administering to said subject a compound selected from thegroup consisting of: rifampicin, disulfiram, and indomethacin, or apharmaceutically acceptable salt thereof.
 25. A method of treatingamyloidosis in a subject, said method comprising administering to saidsubject an effective amount of a combination of (a) a chelator specificfor copper, and (b) clioquinol; wherein said combination preventsformation of Aβ amyloid, promotes, induces or otherwise facilitatesresolubilization of Aβ deposits, or both.
 26. The method of claim 25wherein the chelator specific for copper is specific for the reducedform of copper.
 27. The method of claim 26 wherein the chelator isbathocuproine or a hydrophobic derivative thereof.
 28. A method oftreating amyloidosis in a subject, said method comprising administeringto said subject an effective amount of a combination of (a) analkalinizing agent and (b) clioquinol; wherein said combination preventsformation of Aβ amyloid, promotes, induces or otherwise facilitatesresolubilization of Aβ deposits, or both.
 29. The method of claim 28wherein the alkalinizing agent is magnesium citrate.
 30. The method ofclaim 28 wherein the alkalinizing agent is calcium citrate.
 31. Apharmaceutical composition for treatment of conditions caused byamyloidosis, Aβ-mediated ROS formation, or both, comprising: (a) a metalchelator selected from the group consisting of: bathocuproine,bathophenanthroline, DTPA, EDTA, EGTA, penicillamine, TETA, and TPEN, orhydrophobic derivatives thereof; and (b) clioquinol, together with oneor more pharmaceutically acceptable carriers or diluents.
 32. Thepharmaceutical composition of claim 31 wherein the metal chelator isbathocuproine.
 33. The pharmaceutical composition of claim 31 furthercomprising a supplement selected from the group consisting of: ammoniumsalt, calcium salt, magnesium salt, and sodium salt.
 34. Thepharmaceutical composition of claim 33 wherein the supplement is amagnesium salt.
 35. The pharmaceutical composition of claim 31 furthercomprising a compound selected from the group consisting of: rifampicin,disulfiram, and indomethacin.
 36. A pharmaceutical composition fortreatment of conditions caused by amyloidosis, Aβ-mediated ROSformation, or both, comprising a combination of (a) a salt of a metalchelator selected from the group consisting of: bathocuproine,bathophenanthroline, DTPA, EDTA, EGTA, penicillamine, TETA, and TPEN, orhydrophobic derivatives thereof; and (b) clioquinol; wherein said saltof the metal chelator is selected from the group consisting of:ammonium, calcium, magnesium, and sodium, together with one or morepharmaceutically acceptable carriers or diluents.
 37. The pharmaceuticalcomposition of claim 36 wherein the metal chelator is bathocuproine. 38.The pharmaceutical composition of claim 36 wherein the salt of the metalchelator is a magnesium salt.
 39. The pharmaceutical composition ofclaim 36 further comprising a compound selected from the groupconsisting of: rifampicin, disulfiram, and indomethacin.
 40. Apharmaceutical composition for treatment of conditions caused byamyloidosis, Aβ-mediated ROS formation, or both, comprising a chelatorspecific for copper, with one or more pharmaceutically acceptablecarriers or diluents.
 41. The pharmaceutical composition of claim 40wherein the chelator is specific for the reduced form of copper.
 42. Thepharmaceutical composition of claim 41 wherein the chelator specific forthe reduced form of copper is bathocuproine or a hydrophobic derivativethereof.
 43. A pharmaceutical composition for treatment of conditionscaused by amyloidosis, Aβ-mediated ROS formation, or both, comprising acombination of (a) an alkalinizing agent and (b) clioquinol; togetherwith one or more pharmaceutically acceptable carriers or diluents. 44.The pharmaceutical composition of claim 43 wherein the alkalinizingagent is magnesium citrate.
 45. The pharmaceutical composition of claim43 wherein the alkalinizing agent is calcium citrate.
 46. A compositionof matter comprising: (a) a metal chelator selected from the groupconsisting of: bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA,penicillamine, TETA, and TPEN, or hydrophobic derivatives thereof; and(b) clioquinol.
 47. The composition of claim 46 wherein the metalchelator is bathocuproine.
 48. The composition of claim 46 furthercomprising a supplement selected from the group consisting of: ammoniumsalt, calcium salt, magnesium salt, and sodium salt.
 49. The compositionof claim 48 wherein the supplement is a magnesium salt.
 50. Thecomposition of claim 46 further comprising a compound selected from thegroup consisting of: rifampicin, disulfiram, and indomethacin.
 51. Acomposition of matter comprising a combination of (a) a salt of a metalchelator selected from the group consisting of: bathocuproine,bathophenanthroline, DTPA, EDTA, EGTA, penicillamine, TETA, and TPEN, orhydrophobic derivatives thereof; and (b) clioquinol; wherein said saltof the metal chelator is selected from the group consisting of:ammonium, calcium, magnesium, and sodium.
 52. The composition of claim51 wherein the metal chelator is bathocuproine.
 53. The composition ofclaim 51 wherein the salt of the chelator is a magnesium salt.
 54. Thecomposition of claim 51 further comprising a compound selected from thegroup consisting of: rifampicin, disulfiram, and indomethacin.
 55. Acomposition of matter comprising a combination of (a) an alkalinizingagent and (b) clioquinol.
 56. The composition of claim 55 wherein thealkalinizing agent is magnesium citrate.
 57. The composition of claim 55wherein the alkalinizing agent is calcium citrate.